Sample indexing for single cells

ABSTRACT

Disclosed herein include systems, methods, compositions, and kits for sample identification. A sample indexing composition can comprise, for example, a protein binding reagent associated with a sample indexing oligonucleotide. Different sample indexing compositions can include sample indexing oligonucleotides with different sequences. Sample origin of cells can be identified based on the sequences of the sample indexing oligonucleotides. Sample indexing oligonucleotides can be barcoded using barcoded and lengthened using daisy-chaining primers.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/515,285, filed on Jun. 5, 2017; U.S.Provisional Application No. 62/532,905, filed on Jul. 14, 2017; U.S.Provisional Application No. 62/532,949, filed on Jul. 14, 2017; U.S.Provisional Application No. 62/532,971, filed on Jul. 14, 2017; U.S.Provisional Application No. 62/554,425, filed on Sep. 5, 2017; U.S.Provisional Application No. 62/578,957, filed on Oct. 30, 2017; and U.S.Provisional Application No. 62/645,703, filed on Mar. 20, 2018. Thecontent of each of these related applications is incorporated herein byreference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSequence_Listing_BDCRI_033A.txt, created Mar. 20, 2018, which is 3,180bytes in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

BACKGROUND Field

The present disclosure relates generally to the field of molecularbiology, for example identifying cells of different samples anddetecting interactions between cellular components using molecularbarcoding.

Description of the Related Art

Current technology allows measurement of gene expression of single cellsin a massively parallel manner (e.g., >10000 cells) by attaching cellspecific oligonucleotide barcodes to poly(A) mRNA molecules fromindividual cells as each of the cells is co-localized with a barcodedreagent bead in a compartment. Gene expression may affect proteinexpression. Protein-protein interaction may affect gene expression andprotein expression. There is a need for systems and methods that canquantitatively analyze protein expression, simultaneously measureprotein expression and gene expression in cells, and determiningprotein-protein interactions in cells.

SUMMARY

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein the of the one ormore cells comprises one or more antigen targets, wherein at least onesample indexing composition of the plurality of sample indexingcompositions comprises two or more antigen binding reagents (e.g.,protein binding reagents and antibodies), wherein each of the two ormore antigen binding reagents is associated with a sample indexingoligonucleotide, wherein at least one of the two or more antigen bindingreagents is capable of specifically binding to at least one of the oneor more antigen targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; barcodingthe sample indexing oligonucleotides using a plurality of barcodes tocreate a plurality of barcoded sample indexing oligonucleotides;obtaining sequencing data of the plurality of barcoded sample indexingoligonucleotides; and identifying sample origin of at least one cell ofthe one or more cells based on the sample indexing sequence of at leastone barcoded sample indexing oligonucleotide of the plurality ofbarcoded sample indexing oligonucleotides (e.g., identifying sampleorigin of the plurality of barcoded targets based on the sample indexingsequence of the at least one barcoded sample indexing oligonucleotide).The method can, for example, comprise removing unbound sample indexingcompositions of the plurality of sample indexing compositions.

In some embodiments, the sample indexing sequence is 25-60 nucleotidesin length (e.g., 45 nucleotides in length), about 128 nucleotides inlength, or at least 128 nucleotides in length. Sample indexing sequencesof at least 10 sample indexing compositions of the plurality of sampleindexing compositions can comprise different sequences. Sample indexingsequences of at least 100 or 1000 sample indexing compositions of theplurality of sample indexing compositions can comprise differentsequences.

In some embodiments, the antigen binding reagent comprises an antibody,a tetramer, an aptamers, a protein scaffold, or a combination thereof.The sample indexing oligonucleotide can be conjugated to the antigenbinding reagent through a linker. The oligonucleotide can comprise thelinker. The linker can comprise a chemical group. The chemical group canbe reversibly or irreversibly attached to the antigen binding reagent.The chemical group can be selected from the group consisting of a UVphotocleavable group, a disulfide bond, a streptavidin, a biotin, anamine, and any combination thereof.

In some embodiments, at least one sample of the plurality of samplescomprises a single cell. The at least one of the one or more antigentargets can be on a cell surface or inside of a cell. A sample of theplurality of samples can comprise a plurality of cells, a tissue, atumor sample, or any combination thereof. The plurality of samples cancomprise a mammalian sample, a bacterial sample, a viral sample, a yeastsample, a fungal sample, or any combination thereof.

In some embodiments, removing the unbound sample indexing compositionscomprises washing the one or more cells from each of the plurality ofsamples with a washing buffer. The method can comprise lysing the one ormore cells from each of the plurality of samples. The sample indexingoligonucleotide can be configured to be detachable or non-detachablefrom the antigen binding reagent. The method can comprise detaching thesample indexing oligonucleotide from the antigen binding reagent.Detaching the sample indexing oligonucleotide can comprise detaching thesample indexing oligonucleotide from the antigen binding reagent by UVphotocleaving, chemical treatment (e.g., using a reducing reagent, suchas dithiothreitol), heating, enzyme treatment, or any combinationthereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of the cells of the plurality ofsamples. The sample indexing oligonucleotide can comprise a molecularlabel sequence, a poly(A) region, or a combination thereof. The sampleindexing oligonucleotide can comprise a sequence complementary to acapture sequence of at least one barcode of the plurality of barcodes. Atarget binding region of the barcode can comprise the capture sequence.The target binding region can comprise a poly(dT) region. The sequenceof the sample indexing oligonucleotide complementary to the capturesequence of the barcode can comprise a poly(A) tail. The sample indexingoligonucleotide can comprise a molecular label.

In some embodiments, the antigen target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. The antigen target can be, or comprise, a cell-surface protein,a cell marker, a B-cell receptor, a T-cell receptor, a majorhistocompatibility complex, a tumor antigen, a receptor, an integrin, orany combination thereof. The antigen target can be, or comprise, alipid, a carbohydrate, or any combination thereof. The antigen targetcan be selected from a group comprising 10-100 different antigentargets. The antigen binding reagent can be associated with two or moresample indexing oligonucleotides with an identical sequence. The antigenbinding reagent can be associated with two or more sample indexingoligonucleotides with different sample indexing sequences. The sampleindexing composition of the plurality of sample indexing compositionscan comprise a second antigen binding reagent not conjugated with thesample indexing oligonucleotide. The antigen binding reagent and thesecond antigen binding reagent can be identical.

In some embodiments, a barcode of the plurality of barcodes comprises atarget binding region and a molecular label sequence. Molecular labelsequences of at least two barcodes of the plurality of barcodes comprisedifferent molecule label sequences. The barcode can comprise a celllabel, a binding site for a universal primer, or any combinationthereof. The target binding region can comprise a poly(dT) region.

In some embodiments, the plurality of barcodes is associated with aparticle. At least one barcode of the plurality of barcodes can beimmobilized on the particle, partially immobilized on the particle,enclosed in the particle, partially enclosed in the particle, or anycombination thereof. The particle can be degradable. The particle can bea bead. The bead can be selected from the group consisting ofstreptavidin beads, agarose beads, magnetic beads, conjugated beads,protein A conjugated beads, protein G conjugated beads, protein A/Gconjugated beads, protein L conjugated beads, oligo(dT) conjugatedbeads, silica beads, silica-like beads, anti-biotin microbead,anti-fluorochrome microbead, and any combination thereof. The particlecan comprise a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone,and any combination thereof. The particle can comprise at least 10000barcodes. In some embodiments, the barcodes of the particle can comprisemolecular label sequences selected from at least 1000 or 10000 differentmolecular label sequences. The molecular label sequences of the barcodescan comprise random sequences.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises amplifying the plurality ofbarcoded sample indexing oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded sample indexingoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR) at least a portion of the molecular label sequence and atleast a portion of the sample indexing oligonucleotide. Obtaining thesequencing data of the plurality of barcoded sample indexingoligonucleotides can comprise obtaining sequencing data of the pluralityof amplicons. Obtaining the sequencing data can comprise sequencing atleast a portion of the molecular label sequence and at least a portionof the sample indexing oligonucleotide.

In some embodiments, identifying the sample origin of the at least onecell can comprise identifying sample origin of the plurality of barcodedtargets based on the sample indexing sequence of the at least onebarcoded sample indexing oligonucleotide. Barcoding the sample indexingoligonucleotides using the plurality of barcodes to create the pluralityof barcoded sample indexing oligonucleotides can comprise stochasticallybarcoding the sample indexing oligonucleotides using a plurality ofstochastic barcodes to create a plurality of stochastically barcodedsample indexing oligonucleotides.

In some embodiments, the method comprises: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label, and wherein at least two barcodes of theplurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can comprise: contacting copies of thetargets with target binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets. Prior to obtainingthe sequencing data of the plurality of barcoded targets, the method cancomprise amplifying the barcoded targets to create a plurality ofamplified barcoded targets. Amplifying the barcoded targets to generatethe plurality of amplified barcoded targets can comprise: amplifying thebarcoded targets by polymerase chain reaction (PCR). Barcoding theplurality of targets of the cell using the plurality of barcodes tocreate the plurality of barcoded targets can comprise stochasticallybarcoding the plurality of targets of the cell using a plurality ofstochastic barcodes to create a plurality of stochastically barcodedtargets.

In some embodiments, each of the plurality of sample indexingcompositions comprises the antigen binding reagent. The sample indexingsequences of the sample indexing oligonucleotides associated with thetwo or more antigen binding reagents can be identical. The sampleindexing sequences of the sample indexing oligonucleotides associatedwith the two or more antigen binding reagents can comprise differentsequences. Each of the plurality of sample indexing compositions cancomprise the two or more antigen binding reagents.

Disclosed herein include methods for sample identification. In someembodiments, the method comprise: contacting one or more cells from eachof a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more cellular component targets, wherein atleast one sample indexing composition of the plurality of sampleindexing compositions comprises two or more cellular component bindingreagents (e.g., antigen binding reagents or antibodies), wherein each ofthe two or more cellular component binding reagents is associated with asample indexing oligonucleotide, wherein at least one of the two or morecellular component binding reagents is capable of specifically bindingto at least one of the one or more cellular component targets, whereinthe sample indexing oligonucleotide comprises a sample indexingsequence, and wherein sample indexing sequences of at least two sampleindexing compositions of the plurality of sample indexing compositionscomprise different sequences; barcoding the sample indexingoligonucleotides using a plurality of barcodes to create a plurality ofbarcoded sample indexing oligonucleotides; obtaining sequencing data ofthe plurality of barcoded sample indexing oligonucleotides; andidentifying sample origin of at least one cell of the one or more cellsbased on the sample indexing sequence of at least one barcoded sampleindexing oligonucleotide of the plurality of barcoded sample indexingoligonucleotides. The method can comprise removing unbound sampleindexing compositions of the plurality of sample indexing compositions.

In some embodiments, the sample indexing sequence is 25-60 nucleotidesin length (e.g., 45 nucleotides in length), about 128 nucleotides inlength, or at least 128 nucleotides in length. Sample indexing sequencesof at least 10 sample indexing compositions of the plurality of sampleindexing compositions can comprise different sequences. Sample indexingsequences of at least 10 sample indexing compositions of the pluralityof sample indexing compositions can comprise different sequences. Sampleindexing sequences of at least 10 sample indexing compositions of theplurality of sample indexing compositions comprise different sequences.

In some embodiments, the cellular component binding reagent comprises acell surface binding reagent, an antibody, a tetramer, an aptamers, aprotein scaffold, an integrin, or a combination thereof. The sampleindexing oligonucleotide can be conjugated to the cellular componentbinding reagent through a linker. The oligonucleotide can comprise thelinker. The linker can comprise a chemical group. The chemical group canbe reversibly or irreversibly attached to the cellular component bindingreagent. The chemical group can be selected from the group consisting ofa UV photocleavable group, a disulfide bond, a streptavidin, a biotin,an amine, and any combination thereof.

In some embodiments, at least one sample of the plurality of samplescomprises a single cell. The at least one of the one or more cellularcomponent targets can be expressed on a cell surface. A sample of theplurality of samples can comprise a plurality of cells, a tissue, atumor sample, or any combination thereof. The plurality of samples cancomprise a mammalian sample, a bacterial sample, a viral sample, a yeastsample, a fungal sample, or any combination thereof.

In some embodiments, removing the unbound sample indexing compositionscomprises washing the one or more cells from each of the plurality ofsamples with a washing buffer. The method can comprise lysing the one ormore cells from each of the plurality of samples. The sample indexingoligonucleotide can be configured to be detachable or non-detachablefrom the cellular component binding reagent. The method can comprisedetaching the sample indexing oligonucleotide from the cellularcomponent binding reagent. Detaching the sample indexing oligonucleotidecan comprise detaching the sample indexing oligonucleotide from thecellular component binding reagent by UV photocleaving, chemicaltreatment (e.g., using a reducing reagent, such as dithiothreitol),heating, enzyme treatment, or any combination thereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of the cells of the plurality ofsamples. The sample indexing oligonucleotide can comprise a molecularlabel sequence, a poly(A) region, or a combination thereof. The sampleindexing oligonucleotide can comprise a sequence complementary to acapture sequence of at least one barcode of the plurality of barcodes. Atarget binding region of the barcode can comprise the capture sequence.The target binding region can comprise a poly(dT) region. The sequenceof the sample indexing oligonucleotide complementary to the capturesequence of the barcode can comprise a poly(A) tail. The sample indexingoligonucleotide can comprise a molecular label.

In some embodiments, the cellular component target is, or comprises, acarbohydrate, a lipid, a protein, an extracellular protein, acell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, an intracellular protein, or any combinationthereof. The cellular component target can be selected from a groupcomprising 10-100 different cellular component targets. The cellularcomponent binding reagent can be associated with two or more sampleindexing oligonucleotides with an identical sequence. The cellularcomponent binding reagent can be associated with two or more sampleindexing oligonucleotides with different sample indexing sequences. Thesample indexing composition of the plurality of sample indexingcompositions can comprise a second cellular component binding reagentnot conjugated with the sample indexing oligonucleotide. The cellularcomponent binding reagent and the second cell binding reagent can beidentical.

In some embodiments, a barcode of the plurality of barcodes comprises atarget binding region and a molecular label sequence. Molecular labelsequences of at least two barcodes of the plurality of barcodes cancomprise different molecule label sequences. The barcode can comprise acell label, a binding site for a universal primer, or any combinationthereof. The target binding region can comprise a poly(dT) region.

In some embodiments, the plurality of barcodes is enclosed in aparticle. The particle can be a bead. At least one barcode of theplurality of barcodes can be immobilized on the particle, partiallyimmobilized on the particle, enclosed in the particle, partiallyenclosed in the particle, or any combination thereof. The particle canbe degradable. The bead can be selected from the group consisting ofstreptavidin beads, agarose beads, magnetic beads, conjugated beads,protein A conjugated beads, protein G conjugated beads, protein A/Gconjugated beads, protein L conjugated beads, oligo(dT) conjugatedbeads, silica beads, silica-like beads, anti-biotin microbead,anti-fluorochrome microbead, and any combination thereof. The particlecan comprise a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone,and any combination thereof. The particle can comprise at least 10000barcodes. In some embodiments, the barcodes of the particle can comprisemolecular label sequences selected from at least 1000 or 10000 differentmolecular label sequences. The molecular label sequences of the barcodescan comprise random sequences.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises amplifying the plurality ofbarcoded sample indexing oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded sample indexingoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR) at least a portion of the molecular label sequence and atleast a portion of the sample indexing oligonucleotide. Obtaining thesequencing data of the plurality of barcoded sample indexingoligonucleotides can comprise obtaining sequencing data of the pluralityof amplicons. Obtaining the sequencing data can comprise sequencing atleast a portion of the molecular label sequence and at least a portionof the sample indexing oligonucleotide.

In some embodiments, identifying the sample origin of the at least onecell can comprise identifying sample origin of the plurality of barcodedtargets based on the sample indexing sequence of the at least onebarcoded sample indexing oligonucleotide. Barcoding the sample indexingoligonucleotides using the plurality of barcodes to create the pluralityof barcoded sample indexing oligonucleotides can comprise stochasticallybarcoding the sample indexing oligonucleotides using a plurality ofstochastic barcodes to create a plurality of stochastically barcodedsample indexing oligonucleotides.

In some embodiments, the method comprises: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label, and wherein at least two barcodes of theplurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can comprise: contacting copies of thetargets with target binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets. The method cancomprise: prior to obtaining the sequencing data of the plurality ofbarcoded targets, amplifying the barcoded targets to create a pluralityof amplified barcoded targets. Amplifying the barcoded targets togenerate the plurality of amplified barcoded targets can comprise:amplifying the barcoded targets by polymerase chain reaction (PCR).Barcoding the plurality of targets of the cell using the plurality ofbarcodes to create the plurality of barcoded targets can comprisestochastically barcoding the plurality of targets of the cell using aplurality of stochastic barcodes to create a plurality of stochasticallybarcoded targets.

In some embodiments, each of the plurality of sample indexingcompositions comprises the cellular component binding reagent. Thesample indexing sequences of the sample indexing oligonucleotidesassociated with the two or more cellular component binding reagents canbe identical. The sample indexing sequences of the sample indexingoligonucleotides associated with the two or more cellular componentbinding reagents can comprise different sequences. Each of the pluralityof sample indexing compositions can comprise the two or more cellularcomponent binding reagents.

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more cellular component targets, wherein atleast one sample indexing composition of the plurality of sampleindexing compositions comprises two or more cellular component bindingreagents, wherein each of the two or more cellular component bindingreagents is associated with a sample indexing oligonucleotide, whereinat least one of the two or more cellular component binding reagents iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; andidentifying sample origin of at least one cell of the one or more cellsbased on the sample indexing sequence of at least one sample indexingoligonucleotide of the plurality of sample indexing compositions. Themethod can, for example, include removing unbound sample indexingcompositions of the plurality of sample indexing compositions.

In some embodiments, the sample indexing sequence is 25-60 nucleotidesin length (e.g., 45 nucleotides in length), about 128 nucleotides inlength, or at least 128 nucleotides in length. Sample indexing sequencesof at least 10 sample indexing compositions of the plurality of sampleindexing compositions can comprise different sequences. Sample indexingsequences of at least 100 or 1000 sample indexing compositions of theplurality of sample indexing compositions can comprise differentsequences.

In some embodiments, the antigen binding reagent comprises an antibody,a tetramer, an aptamers, a protein scaffold, or a combination thereof.The sample indexing oligonucleotide can be conjugated to the antigenbinding reagent through a linker. The oligonucleotide can comprise thelinker. The linker can comprise a chemical group. The chemical group canbe reversibly or irreversibly attached to the antigen binding reagent.The chemical group can be selected from the group consisting of a UVphotocleavable group, a disulfide bond, a streptavidin, a biotin, anamine, and any combination thereof.

In some embodiments, at least one sample of the plurality of samplescomprises a single cell. The at least one of the one or more antigentargets can be expressed on a cell surface. A sample of the plurality ofsamples can comprise a plurality of cells, a tissue, a tumor sample, orany combination thereof. The plurality of samples can comprise amammalian sample, a bacterial sample, a viral sample, a yeast sample, afungal sample, or any combination thereof.

In some embodiments, removing the unbound sample indexing compositionscomprises washing the one or more cells from each of the plurality ofsamples with a washing buffer. The method can comprise lysing the one ormore cells from each of the plurality of samples. The sample indexingoligonucleotide can be configured to be detachable or non-detachablefrom the antigen binding reagent. The method can comprise detaching thesample indexing oligonucleotide from the antigen binding reagent.Detaching the sample indexing oligonucleotide can comprise detaching thesample indexing oligonucleotide from the antigen binding reagent by UVphotocleaving, chemical treatment (e.g., using a reducing reagent, suchas dithiothreitol), heating, enzyme treatment, or any combinationthereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of the cells of the plurality ofsamples. The sample indexing oligonucleotide can comprise a molecularlabel sequence, a poly(A) region, or a combination thereof. The sampleindexing oligonucleotide can comprise a sequence complementary to acapture sequence of at least one barcode of the plurality of barcodes. Atarget binding region of the barcode can comprise the capture sequence.The target binding region can comprise a poly(dT) region. The sequenceof the sample indexing oligonucleotide complementary to the capturesequence of the barcode can comprise a poly(A) tail. The sample indexingoligonucleotide can comprise a molecular label.

In some embodiments, the antigen target is, or comprises, acarbohydrate, a lipid, a protein, an extracellular protein, acell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an intracellular protein, or any combination thereof. Theantigen target can be selected from a group comprising 10-100 differentantigen targets. The antigen binding reagent can be associated with twoor more sample indexing oligonucleotides with an identical sequence. Theantigen binding reagent can associated with two or more sample indexingoligonucleotides with different sample indexing sequences. The sampleindexing composition of the plurality of sample indexing compositionscan comprise a second antigen binding reagent not conjugated with thesample indexing oligonucleotide. The antigen binding reagent and thesecond antigen binding reagent can be identical.

In some embodiments, identifying the sample origin of the at least onecell comprises: barcoding sample indexing oligonucleotides of theplurality of sample indexing compositions using a plurality of barcodesto create a plurality of barcoded sample indexing oligonucleotides;obtaining sequencing data of the plurality of barcoded sample indexingoligonucleotides; and identifying the sample origin of the cell based onthe sample indexing sequence of at least one barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides.

In some embodiments, a barcode of the plurality of barcodes comprises atarget binding region and a molecular label sequence. Molecular labelsequences of at least two barcodes of the plurality of barcodes cancomprise different molecule label sequences. The barcode can comprise acell label, a binding site for a universal primer, or any combinationthereof. The target binding region can comprise a poly(dT) region.

In some embodiments, the plurality of barcodes is immobilized on aparticle. At least one barcode of the plurality of barcodes can beimmobilized on the particle, partially immobilized on the particle,enclosed in the particle, partially enclosed in the particle, or anycombination thereof. The particle can be degradable. The particle can bea bead. The bead can be selected from the group consisting ofstreptavidin beads, agarose beads, magnetic beads, conjugated beads,protein A conjugated beads, protein G conjugated beads, protein A/Gconjugated beads, protein L conjugated beads, oligo(dT) conjugatedbeads, silica beads, silica-like beads, anti-biotin microbead,anti-fluorochrome microbead, and any combination thereof. The particlecan comprise a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone,and any combination thereof. The particle can comprise at least 10000barcodes. In some embodiments, the barcodes of the particle can comprisemolecular label sequences selected from at least 1000 or 10000 differentmolecular label sequences. The molecular label sequences of the barcodescan comprise random sequences.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes can comprise: contacting the pluralityof barcodes with the sample indexing oligonucleotides to generatebarcodes hybridized to the sample indexing oligonucleotides; andextending the barcodes hybridized to the sample indexingoligonucleotides to generate the plurality of barcoded sample indexingoligonucleotides. Extending the barcodes can comprise extending thebarcodes using a DNA polymerase to generate the plurality of barcodedsample indexing oligonucleotides. Extending the barcodes can compriseextending the barcodes using a reverse transcriptase to generate theplurality of barcoded sample indexing oligonucleotides.

In some embodiments, the method comprises amplifying the plurality ofbarcoded sample indexing oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded sample indexingoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR) at least a portion of the molecular label sequence and atleast a portion of the sample indexing oligonucleotide. Obtaining thesequencing data of the plurality of barcoded sample indexingoligonucleotides can comprise obtaining sequencing data of the pluralityof amplicons. Obtaining the sequencing data can comprise sequencing atleast a portion of the molecular label sequence and at least a portionof the sample indexing oligonucleotide.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes to create the plurality of barcodedsample indexing oligonucleotides comprises stochastically barcoding thesample indexing oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded sampleindexing oligonucleotides. Identifying the sample origin of the at leastone cell can comprise identifying the presence or absence of the sampleindexing sequence of at least one sample indexing oligonucleotide of theplurality of sample indexing compositions. Identifying the presence orabsence of the sample indexing sequence can comprise: replicating the atleast one sample indexing oligonucleotide to generate a plurality ofreplicated sample indexing oligonucleotides; obtaining sequencing dataof the plurality of replicated sample indexing oligonucleotides; andidentifying the sample origin of the cell based on the sample indexingsequence of a replicated sample indexing oligonucleotide of theplurality of sample indexing oligonucleotides that correspond to theleast one barcoded sample indexing oligonucleotide in the sequencingdata.

In some embodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, ligating a replicating adaptorto the at least one barcoded sample indexing oligonucleotide.Replicating the at least one barcoded sample indexing oligonucleotidecan comprise replicating the at least one barcoded sample indexingoligonucleotide using the replicating adaptor ligated to the at leastone barcoded sample indexing oligonucleotide to generate the pluralityof replicated sample indexing oligonucleotides.

In some embodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, contacting a capture probewith the at least one sample indexing oligonucleotide to generate acapture probe hybridized to the sample indexing oligonucleotide; andextending the capture probe hybridized to the sample indexingoligonucleotide to generate a sample indexing oligonucleotide associatedwith the capture probe. Replicating the at least one sample indexingoligonucleotide can comprise replicating the sample indexingoligonucleotide associated with the capture probe to generate theplurality of replicated sample indexing oligonucleotides.

In some embodiments, the method comprises: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label, and wherein at least two barcodes of theplurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Identifying thesample origin of the at least one barcoded sample indexingoligonucleotide can comprise identifying the sample origin of theplurality of barcoded targets based on the sample indexing sequence ofthe at least one barcoded sample indexing oligonucleotide. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can comprise: contacting copies of thetargets with target binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets. The method cancomprise: prior to obtaining the sequencing data of the plurality ofbarcoded targets, amplifying the barcoded targets to create a pluralityof amplified barcoded targets. Amplifying the barcoded targets togenerate the plurality of amplified barcoded targets can comprise:amplifying the barcoded targets by polymerase chain reaction (PCR).Barcoding the plurality of targets of the cell using the plurality ofbarcodes to create the plurality of barcoded targets can comprisestochastically barcoding the plurality of targets of the cell using aplurality of stochastic barcodes to create a plurality of stochasticallybarcoded targets.

In some embodiments, each of the plurality of sample indexingcompositions comprises the antigen binding reagent. The sample indexingsequences of the sample indexing oligonucleotides associated with thetwo or more antigen binding reagents can be identical. The sampleindexing sequences of the sample indexing oligonucleotides associatedwith the two or more antigen binding reagents can comprise differentsequences. Each of the plurality of sample indexing compositions cancomprise the two or more antigen binding reagents.

Disclosed herein includes a plurality of sample indexing compositions.Each of the plurality of sample indexing compositions can comprise twoor more antigen binding reagents. Each of the two or more antigenbinding reagents can be associated with a sample indexingoligonucleotide. At least one of the two or more antigen bindingreagents can be capable of specifically binding to at least one antigentarget. The sample indexing oligonucleotide can comprise a sampleindexing sequence for identifying sample origin of one or more cells ofa sample. Sample indexing sequences of at least two sample indexingcompositions of the plurality of sample indexing compositions cancomprise different sequences.

In some embodiments, the sample indexing sequence comprises a nucleotidesequence of 25-60 nucleotides in length (e.g., 45 nucleotides inlength), about 128 nucleotides in length, or at least 128 nucleotides inlength. Sample indexing sequences of at least 10, 100, or 1000 sampleindexing compositions of the plurality of sample indexing compositionscomprise different sequences.

In some embodiments, the antigen binding reagent comprises an antibody,a tetramer, an aptamers, a protein scaffold, or a combination thereof.The sample indexing oligonucleotide can be conjugated to the antigenbinding reagent through a linker. The at least one sample indexingoligonucleotide can comprise the linker. The linker can comprise achemical group. The chemical group can be reversibly or irreversiblyattached to the molecule of the antigen binding reagent. The chemicalgroup can be selected from the group consisting of a UV photocleavablegroup, a disulfide bond, a streptavidin, a biotin, an amine, and anycombination thereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of a species. The sample indexingoligonucleotide can comprise a molecular label sequence, a poly(A)region, or a combination thereof. In some embodiments, at least onesample of the plurality of samples can comprise a single cell, aplurality of cells, a tissue, a tumor sample, or any combinationthereof. The sample can comprise a mammalian sample, a bacterial sample,a viral sample, a yeast sample, a fungal sample, or any combinationthereof.

In some embodiments, the antigen target is, or comprises, a cell-surfaceprotein, a cell marker, a B-cell receptor, a T-cell receptor, a majorhistocompatibility complex, a tumor antigen, a receptor, or anycombination thereof. The antigen target can be selected from a groupcomprising 10-100 different antigen targets. The antigen binding reagentcan be associated with two or more sample indexing oligonucleotides withan identical sequence. The antigen binding reagent can be associatedwith two or more sample indexing oligonucleotides with different sampleindexing sequences. The sample indexing composition of the plurality ofsample indexing compositions can comprise a second antigen bindingreagent not conjugated with the sample indexing oligonucleotide. Theantigen binding reagent and the second antigen binding reagent can beidentical.

Disclosed herein include control particle compositions. In someembodiments, the control particle composition comprises a plurality ofcontrol particle oligonucleotides associated with a control particle,wherein each of the plurality of control particle oligonucleotidescomprises a control barcode sequence and a poly(dA) region. At least twoof the plurality of control particle oligonucleotides can comprisedifferent control barcode sequences. The control particleoligonucleotide can comprise a molecular label sequence. The controlparticle oligonucleotide can comprise a binding site for a universalprimer.

In some embodiments, the control barcode sequence is at least 6nucleotides in length, 25-45 nucleotides in length, about 128nucleotides in length, at least 128 nucleotides in length, about 200-500nucleotides in length, or a combination thereof. The control particleoligonucleotide can be about 50 nucleotides in length, about 100nucleotides in length, about 200 nucleotides in length, at least 200nucleotides in length, less than about 200-300 nucleotides in length,about 500 nucleotides in length, or a combination thereof. The controlbarcode sequences of at least 5, 10, 100, 1000, or more of the pluralityof control particle oligonucleotides can be identical. The controlbarcode sequences of about 10, 100, 1000, or more of the plurality ofcontrol particle oligonucleotides can be identical. At least 3, 5, 10,100, or more of the plurality of control particle oligonucleotides cancomprise different control barcode sequences.

In some embodiments, the plurality of control particle oligonucleotidescomprises a plurality of first control particle oligonucleotides eachcomprising a first control barcode sequence, and a plurality of secondcontrol particle oligonucleotides each comprising a second controlbarcode sequence, and wherein the first control barcode sequence and thesecond control barcode sequence have different sequences. The number ofthe plurality of first control particle oligonucleotides and the numberof the plurality of second control particle oligonucleotides can beabout the same. The number of the plurality of first control particleoligonucleotides and the number of the plurality of second controlparticle oligonucleotides can be different. The number of the pluralityof first control particle oligonucleotides can be at least 2 times, 10times, 100 times, or more greater than the number of the plurality ofsecond control particle oligonucleotides.

In some embodiments, the control barcode sequence is not homologous togenomic sequences of a species. The control barcode sequence can behomologous to genomic sequences of a species. The species can be anon-mammalian species. The non-mammalian species can be a phage species.The phage species can be T7 phage, a PhiX phage, or a combinationthereof.

In some embodiments, at least one of the plurality of control particleoligonucleotides is associated with the control particle through alinker. The at least one of the plurality of control particleoligonucleotides can comprise the linker. The linker can comprise achemical group. The chemical group can be reversibly attached to the atleast one of the plurality of control particle oligonucleotides. Thechemical group can comprise a UV photocleavable group, a streptavidin, abiotin, an amine, a disulfide linkage, or any combination thereof.

In some embodiments, the diameter of the control particle is about1-1000 micrometers, about 10-100 micrometers, 7.5 micrometer, or acombination thereof.

In some embodiments, the plurality of control particle oligonucleotidesis immobilized on the control particle. The plurality of controlparticle oligonucleotides can be partially immobilized on the controlparticle. The plurality of control particle oligonucleotides can beenclosed in the control particle. The plurality of control particleoligonucleotides can be partially enclosed in the control particle. Thecontrol particle can be disruptable. The control particle can be a bead.The bead can be, or comprise, a Sepharose bead, a streptavidin bead, anagarose bead, a magnetic bead, a conjugated bead, a protein A conjugatedbead, a protein G conjugated bead, a protein A/G conjugated bead, aprotein L conjugated bead, an oligo(dT) conjugated bead, a silica bead,a silica-like bead, an anti-biotin microbead, an anti-fluorochromemicrobead, or any combination thereof. The control particle can comprisea material of polydimethylsiloxane (PDMS), polystyrene, glass,polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic,plastic, glass, methylstyrene, acrylic polymer, titanium, latex,sepharose, cellulose, nylon, silicone, or any combination thereof. Thecontrol particle can comprise a disruptable hydrogel particle.

In some embodiments, the control particle is associated with adetectable moiety. The control particle oligonucleotide can beassociated with a detectable moiety.

In some embodiments, the control particle is associated with a pluralityof first protein binding reagents, and at least one of the plurality offirst protein binding reagents is associated with one of the pluralityof control particle oligonucleotides. The first protein binding reagentcan comprise a first antibody. The control particle oligonucleotide canbe conjugated to the first protein binding reagent through a linker. Thefirst control particle oligonucleotide can comprise the linker. Thelinker can comprise a chemical group. The chemical group can bereversibly attached to the first protein binding reagent. The chemicalgroup can comprise a UV photocleavable group, a streptavidin, a biotin,an amine, a disulfide linkage, or any combination thereof.

In some embodiments, the first protein binding reagent is associatedwith two or more of the plurality of control particle oligonucleotideswith an identical control barcode sequence. The first protein bindingreagent can be associated with two or more of the plurality of controlparticle oligonucleotides with different control barcode sequences. Insome embodiments, at least one of the plurality of first protein bindingreagents is not associated with any of the plurality of control particleoligonucleotides. The first protein binding reagent associated with thecontrol particle oligonucleotide and the first protein binding reagentnot associated with any control particle oligonucleotide can beidentical protein binding reagents.

In some embodiments, the control particle is associated with a pluralityof second protein binding reagents. At least one of the plurality ofsecond protein binding reagents can be associated with one of theplurality of control particle oligonucleotides. The control particleoligonucleotide associated with the first protein binding reagent andthe control particle oligonucleotide associated with the second proteinbinding reagent can comprise different control barcode sequences. Thefirst protein binding reagent and the second protein binding reagent canbe identical protein binding reagents.

In some embodiments, the first protein binding reagent can be associatedwith a partner binding reagent, and wherein the first protein bindingreagent is associated with the control particle using the partnerbinding reagent. The partner binding reagent can comprise a partnerantibody. The partner antibody can comprise an anti-cat antibody, ananti-chicken antibody, an anti-cow antibody, an anti-dog antibody, ananti-donkey antibody, an anti-goat antibody, an anti-guinea pigantibody, an anti-hamster antibody, an anti-horse antibody, ananti-human antibody, an anti-llama antibody, an anti-monkey antibody, ananti-mouse antibody, an anti-pig antibody, an anti-rabbit antibody, ananti-rat antibody, an anti-sheep antibody, or a combination thereof. Thepartner antibody can comprise an immunoglobulin G (IgG), a F(ab′)fragment, a F(ab′)2 fragment, a combination thereof, or a fragmentthereof.

In some embodiments, the first protein binding reagent can be associatedwith a detectable moiety. The second protein binding reagent can beassociated with a detectable moiety.

Disclosed herein include methods for determining the numbers of targets.In some embodiments, the method comprises: stochastically barcoding aplurality of targets of a cell of a plurality of cells and a pluralityof control particle oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded targets and aplurality of stochastically barcoded control particle oligonucleotides,wherein each of the plurality of stochastic barcodes comprises a celllabel sequence, a molecular label sequence, and a target-binding region,wherein the molecular label sequences of at least two stochasticbarcodes of the plurality of stochastic barcodes comprise differentsequences, and wherein at least two stochastic barcodes of the pluralityof stochastic barcodes comprise an identical cell label sequence,wherein a control particle composition comprises a control particleassociated with the plurality of control particle oligonucleotides,wherein each of the plurality of control particle oligonucleotidescomprises a control barcode sequence and a pseudo-target regioncomprising a sequence substantially complementary to the target-bindingregion of at least one of the plurality of stochastic barcodes. Themethod can comprise: obtaining sequencing data of the plurality ofstochastically barcoded targets and the plurality of stochasticallybarcoded control particle oligonucleotides; counting the number ofmolecular label sequences with distinct sequences associated with theplurality of control particle oligonucleotides with the control barcodesequence in the sequencing data. The method can comprise: for at leastone target of the plurality of targets: counting the number of molecularlabel sequences with distinct sequences associated with the target inthe sequencing data; and estimating the number of the target, whereinthe number of the target estimated correlates with the number ofmolecular label sequences with distinct sequences associated with thetarget counted and the number of molecular label sequences with distinctsequences associated with the control barcode sequence.

In some embodiments, the pseudo-target region comprises a poly(dA)region. The pseudo-target region can comprise a subsequence of thetarget. In some embodiments, the control barcode sequence can be atleast 6 nucleotides in length, 25-45 nucleotides in length, about 128nucleotides in length, at least 128 nucleotides in length, about 200-500nucleotides in length, or a combination thereof. The control particleoligonucleotide can be about 50 nucleotides in length, about 100nucleotides in length, about 200 nucleotides in length, at least 200nucleotides in length, less than about 200-300 nucleotides in length,about 500 nucleotides in length, or any combination thereof. The controlbarcode sequences of at least 5, 10, 100, 1000, or more of the pluralityof control particle oligonucleotides can be identical. At least 3, 5,10, 100, or more of the plurality of control particle oligonucleotidescan comprise different control barcode sequences.

In some embodiments, the plurality of control particle oligonucleotidescomprises a plurality of first control particle oligonucleotides eachcomprising a first control barcode sequence, and a plurality of secondcontrol particle oligonucleotides each comprising a second controlbarcode sequence. The first control barcode sequence and the secondcontrol barcode sequence can have different sequences. The number of theplurality of first control particle oligonucleotides and the number ofthe plurality of second control particle oligonucleotides can be aboutthe same. The number of the plurality of first control particleoligonucleotides and the number of the plurality of second controlparticle oligonucleotides can be different. The number of the pluralityof first control particle oligonucleotides can be at least 2 times, 10times, 100 times, or more greater than the number of the plurality ofsecond control particle oligonucleotides.

In some embodiments, counting the number of molecular label sequenceswith distinct sequences associated with the plurality of controlparticle oligonucleotides with the control barcode sequence in thesequencing data comprises: counting the number of molecular labelsequences with distinct sequences associated with the first controlbarcode sequence in the sequencing data; and counting the number ofmolecular label sequences with distinct sequences associated with thesecond control barcode sequence in the sequencing data. The number ofthe target estimated can correlate with the number of molecular labelsequences with distinct sequences associated with the target counted,the number of molecular label sequences with distinct sequencesassociated with the first control barcode sequence, and the number ofmolecular label sequences with distinct sequences associated with thesecond control barcode sequence. The number of the target estimated cancorrelate with the number of molecular label sequences with distinctsequences associated with the target counted, the number of molecularlabel sequences with distinct sequences associated with the controlbarcode sequence, and the number of the plurality of control particleoligonucleotides comprising the control barcode sequence. The number ofthe target estimated can correlate with the number of molecular labelsequences with distinct sequences associated with the target counted,and a ratio of the number of the plurality of control particleoligonucleotides comprising the control barcode sequence and the numberof molecular label sequences with distinct sequences associated with thecontrol barcode sequence.

In some embodiments, the control particle oligonucleotide is nothomologous to genomic sequences of the cell. The control particleoligonucleotide can be not homologous to genomic sequences of thespecies. The control particle oligonucleotide can be homologous togenomic sequences of a species. The species can be a non-mammalianspecies. The non-mammalian species can be a phage species. The phagespecies can be T7 phage, a PhiX phage, or a combination thereof.

In some embodiments, the control particle oligonucleotide can beconjugated to the control particle through a linker. At least one of theplurality of control particle oligonucleotides can be associated withthe control particle through a linker. The at least one of the pluralityof control particle oligonucleotides can comprise the linker. Thechemical group can be reversibly attached to the at least one of theplurality of control particle oligonucleotides. The chemical group cancomprise a UV photocleavable group, a streptavidin, a biotin, an amine,a disulfide linkage, or any combination thereof.

In some embodiments, the diameter of the control particle is about1-1000 micrometers, about 10-100 micrometers, about 7.5 micrometer, or acombination thereof. The plurality of control particle oligonucleotidesis immobilized on the control particle. The plurality of controlparticle oligonucleotides can be partially immobilized on the controlparticle. The plurality of control particle oligonucleotides can beenclosed in the control particle. The plurality of control particleoligonucleotides can be partially enclosed in the control particle.

In some embodiments, the method comprises releasing the at least one ofthe plurality of control particle oligonucleotides from the controlparticle prior to stochastically barcoding the plurality of targets andthe control particle and the plurality of control particleoligonucleotides.

In some embodiments, the control particle is disruptable. The controlparticle can be a control particle bead. The control particle bead cancomprise a Sepharose bead, a streptavidin bead, an agarose bead, amagnetic bead, a conjugated bead, a protein A conjugated bead, a proteinG conjugated bead, a protein A/G conjugated bead, a protein L conjugatedbead, an oligo(dT) conjugated bead, a silica bead, a silica-like bead,an anti-biotin microbead, an anti-fluorochrome microbead, or anycombination thereof. The control particle can comprise a material ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone,or any combination thereof. The control particle can comprise adisruptable hydrogel particle.

In some embodiments, the control particle is associated with adetectable moiety. The control particle oligonucleotide can beassociated with a detectable moiety.

In some embodiments, the control particle can be associated with aplurality of first protein binding reagents, and at least one of theplurality of first protein binding reagents can be associated with oneof the plurality of control particle oligonucleotides. The first proteinbinding reagent can comprise a first antibody. The control particleoligonucleotide can be conjugated to the first protein binding reagentthrough a linker. The first control particle oligonucleotide cancomprise the linker. The linker can comprise a chemical group. Thechemical group can be reversibly attached to the first protein bindingreagent. The chemical group can comprise a UV photocleavable group, astreptavidin, a biotin, an amine, a disulfide linkage, or anycombination thereof.

In some embodiments, the first protein binding reagent can be associatedwith two or more of the plurality of control particle oligonucleotideswith an identical control barcode sequence. The first protein bindingreagent can be associated with two or more of the plurality of controlparticle oligonucleotides with different control barcode sequences. Atleast one of the plurality of first protein binding reagents can be notassociated with any of the plurality of control particleoligonucleotides. The first protein binding reagent associated with thecontrol particle oligonucleotide and the first protein binding reagentnot associated with any control particle oligonucleotide can beidentical protein binding reagents. The control particle can associatedwith a plurality of second protein binding reagents At least one of theplurality of second protein binding reagents can be associated with oneof the plurality of control particle oligonucleotides. The controlparticle oligonucleotide associated with the first protein bindingreagent and the control particle oligonucleotide associated with thesecond protein binding reagent can comprise different control barcodesequences. The first protein binding reagent and the second proteinbinding reagent can be identical protein binding reagents.

In some embodiments, the first protein binding reagent is associatedwith a partner binding reagent, and wherein the first protein bindingreagent is associated with the control particle using the partnerbinding reagent. The partner binding reagent can comprise a partnerantibody. The partner antibody can comprise an anti-cat antibody, ananti-chicken antibody, an anti-cow antibody, an anti-dog antibody, ananti-donkey antibody, an anti-goat antibody, an anti-guinea pigantibody, an anti-hamster antibody, an anti-horse antibody, ananti-human antibody, an anti-llama antibody, an anti-monkey antibody, ananti-mouse antibody, an anti-pig antibody, an anti-rabbit antibody, ananti-rat antibody, an anti-sheep antibody, or a combination thereof. Thepartner antibody can comprise an immunoglobulin G (IgG), a F(ab′)fragment, a F(ab′)2 fragment, a combination thereof, or a fragmentthereof.

In some embodiments, the first protein binding reagent can be associatedwith a detectable moiety. The second protein binding reagent can beassociated with a detectable moiety.

In some embodiments, the stochastic barcode comprises a binding site fora universal primer. The target-binding region can comprise a poly(dT)region.

In some embodiments, the plurality of stochastic barcodes is associatedwith a barcoding particle. At least one stochastic barcode of theplurality of stochastic barcodes can be immobilized on the barcodingparticle. At least one stochastic barcode of the plurality of stochasticbarcodes can be partially immobilized on the barcoding particle. Atleast one stochastic barcode of the plurality of stochastic barcodes canbe enclosed in the barcoding particle. At least one stochastic barcodeof the plurality of stochastic barcodes can be partially enclosed in thebarcoding particle.

In some embodiments, the barcoding particle is disruptable. Thebarcoding particle can be a barcoding bead. The barcoding bead cancomprise a Sepharose bead, a streptavidin bead, an agarose bead, amagnetic bead, a conjugated bead, a protein A conjugated bead, a proteinG conjugated bead, a protein A/G conjugated bead, a protein L conjugatedbead, an oligo(dT) conjugated bead, a silica bead, a silica-like bead,an anti-biotin microbead, an anti-fluorochrome microbead, or anycombination thereof. The barcoding particle can comprise a material ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone,or any combination thereof. The barcoding particle can comprise adisruptable hydrogel particle.

In some embodiments, the stochastic barcodes of the barcoding particlecomprise molecular label sequences selected from at least 1000, 10000,or more different molecular label sequences. In some embodiments, themolecular label sequences of the stochastic barcodes comprise randomsequences. In some embodiments, The barcoding particle comprises atleast 10000 stochastic barcodes.

In some embodiments, stochastically barcoding the plurality of targetsand the plurality of control particle oligonucleotides using theplurality of stochastic barcodes comprises: contacting the plurality ofstochastic barcodes with targets of the plurality of targets and controlparticle oligonucleotides of the plurality of control particleoligonucleotides to generate stochastic barcodes hybridized to thetargets and the control particle oligonucleotides; and extending thestochastic barcodes hybridized to the targets and the control particleoligonucleotides to generate the plurality of stochastically barcodedtargets and the plurality of stochastically barcoded control particleoligonucleotides. Extending the stochastic barcodes can compriseextending the stochastic barcodes using a DNA polymerase, a reversetranscriptase, or a combination thereof.

In some embodiments, the method comprises amplifying the plurality ofstochastically barcoded targets and the plurality of stochasticallybarcoded control particle oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of stochastically barcoded targetsand the plurality of stochastically barcoded control particleoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of the molecular label sequence andat least a portion of the control particle oligonucleotide or at least aportion of the molecular label sequence and at least a portion of thecontrol particle oligonucleotide. Obtaining the sequencing data cancomprise obtaining sequencing data of the plurality of amplicons.Obtaining the sequencing data can comprise sequencing the at least aportion of the molecular label sequence and the at least a portion ofthe control particle oligonucleotide, or the at least a portion of themolecular label sequence and the at least a portion of the controlparticle oligonucleotide.

Disclosed herein also include kits for sequencing control. In someembodiments, the kit comprises: a control particle compositioncomprising a plurality of control particle oligonucleotides associatedwith a control particle, wherein each of the plurality of controlparticle oligonucleotides comprises a control barcode sequence and apoly(dA) region.

In some embodiments, at least two of the plurality of control particleoligonucleotides comprise different control barcode sequences. In someembodiments, the control barcode sequence can be at least 6 nucleotidesin length, 25-45 nucleotides in length, about 128 nucleotides in length,at least 128 nucleotides in length, about 200-500 nucleotides in length,or a combination thereof. The control particle oligonucleotide can beabout 50 nucleotides in length, about 100 nucleotides in length, about200 nucleotides in length, at least 200 nucleotides in length, less thanabout 200-300 nucleotides in length, about 500 nucleotides in length, orany combination thereof. The control barcode sequences of at least 5,10, 100, 1000, or more of the plurality of control particleoligonucleotides can be identical. At least 3, 5, 10, 100, or more ofthe plurality of control particle oligonucleotides can comprisedifferent control barcode sequences.

In some embodiments, the plurality of control particle oligonucleotidescomprises a plurality of first control particle oligonucleotides eachcomprising a first control barcode sequence, and a plurality of secondcontrol particle oligonucleotides each comprising a second controlbarcode sequence. The first control barcode sequence and the secondcontrol barcode sequence can have different sequences. The number of theplurality of first control particle oligonucleotides and the number ofthe plurality of second control particle oligonucleotides can be aboutthe same. The number of the plurality of first control particleoligonucleotides and the number of the plurality of second controlparticle oligonucleotides can be different. The number of the pluralityof first control particle oligonucleotides can be at least 2 times, 10times, 100 times, or more greater than the number of the plurality ofsecond control particle oligonucleotides.

In some embodiments, the control particle oligonucleotide is nothomologous to genomic sequences of the cell. The control particleoligonucleotide can be not homologous to genomic sequences of thespecies. The control particle oligonucleotide can be homologous togenomic sequences of a species. The species can be a non-mammalianspecies. The non-mammalian species can be a phage species. The phagespecies can be T7 phage, a PhiX phage, or a combination thereof.

In some embodiments, the control particle oligonucleotide can beconjugated to the control particle through a linker. At least one of theplurality of control particle oligonucleotides can be associated withthe control particle through a linker. The at least one of the pluralityof control particle oligonucleotides can comprise the linker. Thechemical group can be reversibly attached to the at least one of theplurality of control particle oligonucleotides. The chemical group cancomprise a UV photocleavable group, a streptavidin, a biotin, an amine,a disulfide linkage, or any combination thereof.

In some embodiments, the diameter of the control particle is about1-1000 micrometers, about 10-100 micrometers, about 7.5 micrometer, or acombination thereof. The plurality of control particle oligonucleotidesis immobilized on the control particle. The plurality of controlparticle oligonucleotides can be partially immobilized on the controlparticle. The plurality of control particle oligonucleotides can beenclosed in the control particle. The plurality of control particleoligonucleotides can be partially enclosed in the control particle.

In some embodiments, the kit comprises a plurality of barcodes. Abarcode of the plurality of barcodes can comprise a target-bindingregion and a molecular label sequence, and molecular label sequences ofat least two barcodes of the plurality of barcodes can comprisedifferent molecule label sequences. The barcode can comprise a celllabel sequence, a binding site for a universal primer, or anycombination thereof. The target-binding region comprises a poly(dT)region.

In some embodiments, the plurality of barcodes can be associated with abarcoding particle. At least one barcode of the plurality of barcodescan be immobilized on the barcoding particle. At least one barcode ofthe plurality of barcodes is partially immobilized on the barcodingparticle. At least one barcode of the plurality of barcodes can beenclosed in the barcoding particle. At least one barcode of theplurality of barcodes can be partially enclosed in the barcodingparticle. The barcoding particle can be disruptable. The barcodingparticle can be a second bead. The bead can be, or comprise, a Sepharosebead, a streptavidin bead, an agarose bead, a magnetic bead, aconjugated bead, a protein A conjugated bead, a protein G conjugatedbead, a protein A/G conjugated bead, a protein L conjugated bead, anoligo(dT) conjugated bead, a silica bead, a silica-like bead, ananti-biotin microbead, an anti-fluorochrome microbead, or anycombination thereof. The barcoding particle can comprise a materialselected from the group consisting of polydimethylsiloxane (PDMS),polystyrene, glass, polypropylene, agarose, gelatin, hydrogel,paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer,titanium, latex, sepharose, cellulose, nylon, silicone, and anycombination thereof. The barcoding particle can comprise a disruptablehydrogel particle.

In some embodiments, the barcodes of the barcoding particle comprisemolecular label sequences selected from at least 1000, 10000, or moredifferent molecular label sequences. The molecular label sequences ofthe barcodes can comprise random sequences. The barcoding particle cancomprise at least 10000 barcodes. The kit can comprise a DNA polymerase.The kit can comprise reagents for polymerase chain reaction (PCR).

Methods disclosed herein for cell identification can comprise:contacting a first plurality of cells and a second plurality of cellswith two sample indexing compositions respectively, wherein each of thefirst plurality of cells and each of the second plurality of cellscomprises one or more protein targets, wherein each of the two sampleindexing compositions comprises a protein binding reagent associatedwith a sample indexing oligonucleotide, wherein the protein bindingreagent is capable of specifically binding to at least one of the one ormore protein targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of the two sample indexing compositions comprise differentsequences; barcoding the sample indexing oligonucleotides using aplurality of barcodes to create a plurality of barcoded sample indexingoligonucleotides, wherein each of the plurality of barcodes comprises acell label sequence, a molecular label sequence, and a target-bindingregion, wherein the molecular label sequences of at least two barcodesof the plurality of barcodes comprise different sequences, and whereinat least two barcodes of the plurality of barcodes comprise an identicalcell label sequence; obtaining sequencing data of the plurality ofbarcoded sample indexing oligonucleotides; and identifying a cell labelsequence associated with two or more sample indexing sequences in thesequencing data obtained; and removing sequencing data associated withthe cell label sequence from the sequencing data obtained. In someembodiments, the sample indexing oligonucleotide comprises a molecularlabel sequence, a binding site for a universal primer, or a combinationthereof. As described herein, the first plurality of cells can beobtained or derived from a different tissue or organ than the secondplurality of cells, and the first plurality of cells and the secondplurality of cells can be from the same or different subjects (e.g., amammal). For example, the first plurality of cells can be obtained orderived from a different human subject than the second plurality ofcells. In some embodiments, the first plurality of cells and the secondplurality of cells are obtained or derived from different tissues of thesame human subject.

In some embodiments, contacting the first plurality of cells and thesecond plurality of cells with the two sample indexing compositionsrespectively comprises: contacting the first plurality of cells with afirst sample indexing compositions of the two sample indexingcompositions; and contacting the first plurality of cells with a secondsample indexing compositions of the two sample indexing compositions.

As described herein, the sample indexing sequence can be, for example,at least 6 nucleotides in length, 25-45 nucleotides in length, about 128nucleotides in length, at least 128 nucleotides in length, about 200-500nucleotides in length, or a combination thereof. The sample indexingoligonucleotide can be about 50 nucleotides in length, about 100nucleotides in length, about 200 nucleotides in length, at least 200nucleotides in length, less than about 200-300 nucleotides in length,about 500 nucleotides in length, or a combination thereof. In someembodiments, sample indexing sequences of at least 10, 100, 1000, ormore sample indexing compositions of the plurality of sample indexingcompositions comprise different sequences.

In some embodiments, the protein binding reagent comprises an antibody,a tetramer, an aptamers, a protein scaffold, or a combination thereof.The sample indexing oligonucleotide can be conjugated to the proteinbinding reagent through a linker. The oligonucleotide can comprise thelinker. The linker can comprise a chemical group. The chemical group canbe reversibly attached to the protein binding reagent. The chemicalgroup can comprise a UV photocleavable group, a streptavidin, a biotin,an amine, a disulfide linkage or any combination thereof.

In some embodiments, at least one sample of the plurality of samplescomprises a single cell. The at least one of the one or more proteintargets can be on a cell surface.

In some embodiments, the method comprises: removing unbound sampleindexing compositions of the two sample indexing compositions. Removingthe unbound sample indexing compositions can comprise washing cells ofthe first plurality of cells and the second plurality of cells with awashing buffer. Removing the unbound sample indexing compositions cancomprise selecting cells bound to at least one protein binding reagentof the two sample indexing compositions using flow cytometry. In someembodiments, the method comprises: lysing the one or more cells fromeach of the plurality of samples.

In some embodiments, the sample indexing oligonucleotide is configuredto be detachable or non-detachable from the protein binding reagent. Themethod can comprise detaching the sample indexing oligonucleotide fromthe protein binding reagent. Detaching the sample indexingoligonucleotide can comprise detaching the sample indexingoligonucleotide from the protein binding reagent by UV photocleaving,chemical treatment, heating, enzyme treatment, or any combinationthereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of any of the one or more cells. Thecontrol barcode sequence may be not homologous to genomic sequences of aspecies. The species can be a non-mammalian species. The non-mammalianspecies can be a phage species. The phage species is T7 phage, a PhiXphage, or a combination thereof.

In some embodiments, a sample of the plurality of samples comprises aplurality of cells, a tissue, a tumor sample, or any combinationthereof. The plurality of sample can comprise a mammalian cell, abacterial cell, a viral cell, a yeast cell, a fungal cell, or anycombination thereof. The sample indexing oligonucleotide can comprise asequence complementary to a capture sequence of at least one barcode ofthe plurality of barcodes. The barcode can comprise a target-bindingregion which comprises the capture sequence. The target-binding regioncan comprise a poly(dT) region. The sequence of the sample indexingoligonucleotide complementary to the capture sequence of the barcode cancomprise a poly(dA) region.

In some embodiments, the protein target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. The protein target can be, or comprise, a cell-surface protein,a cell marker, a B-cell receptor, a T-cell receptor, a majorhistocompatibility complex, a tumor antigen, a receptor, an integrin, orany combination thereof. The protein target can be, or comprise, alipid, a carbohydrate, or any combination thereof. The protein targetcan be selected from a group comprising 10-100 different proteintargets.

In some embodiments, the protein binding reagent is associated with twoor more sample indexing oligonucleotides with an identical sequence. Theprotein binding reagent can be associated with two or more sampleindexing oligonucleotides with different sample indexing sequences. Thesample indexing composition of the plurality of sample indexingcompositions can comprise a second protein binding reagent notconjugated with the sample indexing oligonucleotide. The protein bindingreagent and the second protein binding reagent can be identical.

In some embodiments, a barcode of the plurality of barcodes comprises atarget-binding region and a molecular label sequence, and molecularlabel sequences of at least two barcodes of the plurality of barcodescomprise different molecule label sequences. The barcode can comprise acell label sequence, a binding site for a universal primer, or anycombination thereof. The target-binding region can comprise a poly(dT)region.

In some embodiments, the plurality of barcodes can be associated with aparticle. At least one barcode of the plurality of barcodes can beimmobilized on the particle. At least one barcode of the plurality ofbarcodes can be partially immobilized on the particle. At least onebarcode of the plurality of barcodes can be enclosed in the particle. Atleast one barcode of the plurality of barcodes can be partially enclosedin the particle. The particle can be disruptable. The particle can be abead. The bead can be, or comprise, a Sepharose bead, a streptavidinbead, an agarose bead, a magnetic bead, a conjugated bead, a protein Aconjugated bead, a protein G conjugated bead, a protein A/G conjugatedbead, a protein L conjugated bead, an oligo(dT) conjugated bead, asilica bead, a silica-like bead, an anti-biotin microbead, ananti-fluorochrome microbead, or any combination thereof. The particlecan comprise a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone,and any combination thereof. The particle can comprise a disruptablehydrogel particle.

In some embodiments, the protein binding reagent is associated with adetectable moiety. In some embodiments, the particle is associated witha detectable moiety. The sample indexing oligonucleotide is associatedwith an optical moiety.

In some embodiments, the barcodes of the particle can comprise molecularlabel sequences selected from at least 1000, 10000, or more differentmolecular label sequences. The molecular label sequences of the barcodescan comprise random sequences. The particle can comprise at least 10000barcodes.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises: amplifying the plurality ofbarcoded sample indexing oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded sample indexingoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of the molecular label sequence andat least a portion of the sample indexing oligonucleotide. In someembodiments, obtaining the sequencing data of the plurality of barcodedsample indexing oligonucleotides can comprise obtaining sequencing dataof the plurality of amplicons. Obtaining the sequencing data comprisessequencing at least a portion of the molecular label sequence and atleast a portion of the sample indexing oligonucleotide. In someembodiments, identifying the sample origin of the at least one cellcomprises identifying sample origin of the plurality of barcoded targetsbased on the sample indexing sequence of the at least one barcodedsample indexing oligonucleotide.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes to create the plurality of barcodedsample indexing oligonucleotides comprises stochastically barcoding thesample indexing oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label sequence, and wherein at least two barcodes ofthe plurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can comprise: contacting copies of thetargets with target-binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets.

In some embodiments, the method comprises: prior to obtaining thesequencing data of the plurality of barcoded targets, amplifying thebarcoded targets to create a plurality of amplified barcoded targets.Amplifying the barcoded targets to generate the plurality of amplifiedbarcoded targets can comprise: amplifying the barcoded targets bypolymerase chain reaction (PCR). Barcoding the plurality of targets ofthe cell using the plurality of barcodes to create the plurality ofbarcoded targets can comprise stochastically barcoding the plurality oftargets of the cell using a plurality of stochastic barcodes to create aplurality of stochastically barcoded targets.

Also disclosed herein include methods and compositions that can be usedfor sequencing control. In some embodiments, the method for sequencingcontrol comprises: contacting one or more cells of a plurality of cellswith a control composition of a plurality of control compositions,wherein a cell of the plurality of cells comprises a plurality oftargets and a plurality of protein targets, wherein each of theplurality of control compositions comprises a protein binding reagentassociated with a control oligonucleotide, wherein the protein bindingreagent is capable of specifically binding to at least one of theplurality of protein targets, and wherein the control oligonucleotidecomprises a control barcode sequence and a pseudo-target regioncomprising a sequence substantially complementary to the target-bindingregion of at least one of the plurality of barcodes; barcoding thecontrol oligonucleotides using a plurality of barcodes to create aplurality of barcoded control oligonucleotides, wherein each of theplurality of barcodes comprises a cell label sequence, a molecular labelsequence, and a target-binding region, wherein the molecular labelsequences of at least two barcodes of the plurality of barcodes comprisedifferent sequences, and wherein at least two barcodes of the pluralityof barcodes comprise an identical cell label sequence; obtainingsequencing data of the plurality of barcoded control oligonucleotides;determining at least one characteristic of the one or more cells usingat least one characteristic of the plurality of barcoded controloligonucleotides in the sequencing data. In some embodiments, thepseudo-target region comprises a poly(dA) region.

In some embodiments, the control barcode sequence is at least 6nucleotides in length, 25-45 nucleotides in length, about 128nucleotides in length, at least 128 nucleotides in length, about 200-500nucleotides in length, or a combination thereof. The control particleoligonucleotide can be about 50 nucleotides in length, about 100nucleotides in length, about 200 nucleotides in length, at least 200nucleotides in length, less than about 200-300 nucleotides in length,about 500 nucleotides in length, or a combination thereof. The controlbarcode sequences of at least 2, 10, 100, 1000, or more of the pluralityof control particle oligonucleotides can be identical. At least 2, 10,100, 1000, or more of the plurality of control particle oligonucleotidescan comprise different control barcode sequences.

In some embodiments, determining the at least one characteristic of theone or more cells comprises: determining the number of cell labelsequences with distinct sequences associated with the plurality ofbarcoded control oligonucleotides in the sequencing data; anddetermining the number of the one or more cells using the number of celllabel sequences with distinct sequences associated with the plurality ofbarcoded control oligonucleotides. The method can comprise: determiningsingle cell capture efficiency based the number of the one or more cellsdetermined. The method can comprise: comprising determining single cellcapture efficiency based on the ratio of the number of the one or morecells determined and the number of the plurality of cells.

In some embodiments, determining the at least one characteristic of theone or more cells using the characteristics of the plurality of barcodedcontrol oligonucleotides in the sequencing data comprises: for each celllabel in the sequencing data, determining the number of molecular labelsequences with distinct sequences associated with the cell label and thecontrol barcode sequence; and determining the number of the one or morecells using the number of molecular label sequences with distinctsequences associated with the cell label and the control barcodesequence. Determining the number of molecular label sequences withdistinct sequences associated with the cell label and the controlbarcode sequence can comprise: for each cell label in the sequencingdata, determining the number of molecular label sequences with thehighest number of distinct sequences associated with the cell label andthe control barcode sequence. Determining the number of the one or morecells using the number of molecular label sequences with distinctsequences associated with the cell label and the control barcodesequence can comprise: generating a plot of the number of molecularlabel sequences with the highest number of distinct sequences with thenumber of cell labels in the sequencing data associated with the numberof molecular label sequences with the highest number of distinctsequences; and determining a cutoff in the plot as the number of the oneor more cells.

In some embodiments, the control oligonucleotide is not homologous togenomic sequences of any of the plurality of cells. The controloligonucleotide can be homologous to genomic sequences of a species. Thespecies can be a non-mammalian species. The non-mammalian species can bea phage species. The phage species can be T7 phage, a PhiX phage, or acombination thereof.

In some embodiments, the method comprises releasing the controloligonucleotide from the protein binding reagent prior to barcoding thecontrol oligonucleotides. In some embodiments, the method comprisesremoving unbound control compositions of the plurality of controlcompositions. Removing the unbound control compositions can comprisewashing the one or more cells of the plurality of cells with a washingbuffer. Removing the unbound sample indexing compositions can compriseselecting cells bound to at least one protein binding reagent of thecontrol composition using flow cytometry.

In some embodiments, at least one of the plurality of protein targets ison a cell surface. At least one of the plurality of protein targets cancomprise a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, or any combination thereof. The protein bindingreagent can comprise an antibody. The control oligonucleotide can beconjugated to the protein binding reagent through a linker. The controloligonucleotide can comprise the linker. The linker can comprise achemical group. The chemical group can be reversibly attached to thefirst protein binding reagent. The chemical group can comprise a UVphotocleavable group, a streptavidin, a biotin, an amine, a disulfidelinkage, or any combination thereof.

In some embodiments, the protein binding reagent is associated with twoor more control oligonucleotides with an identical control barcodesequence. The protein binding reagent can be associated with two or morecontrol oligonucleotides with different identical control barcodesequences. In some embodiments, a second protein binding reagent of theplurality of control compositions is not associated with the controloligonucleotide. The protein binding reagent and the second proteinbinding reagent can be identical.

In some embodiments, the barcode comprises a binding site for auniversal primer. The target-binding region can comprise a poly(dT)region. In some embodiments, the plurality of barcodes is associatedwith a barcoding particle. At least one barcode of the plurality ofbarcodes can be immobilized on the barcoding particle. At least onebarcode of the plurality of barcodes can be partially immobilized on thebarcoding particle. At least one barcode of the plurality of barcodes isenclosed in the barcoding particle. At least one barcode of theplurality of barcodes is partially enclosed in the barcoding particle.The barcoding particle can be disruptable. The barcoding particle can bea barcoding bead. The barcoding bead can comprise a Sepharose bead, astreptavidin bead, an agarose bead, a magnetic bead, a conjugated bead,a protein A conjugated bead, a protein G conjugated bead, a protein A/Gconjugated bead, a protein L conjugated bead, an oligo(dT) conjugatedbead, a silica bead, a silica-like bead, an anti-biotin microbead, ananti-fluorochrome microbead, or any combination thereof. The barcodingparticle can comprise a material of polydimethylsiloxane (PDMS),polystyrene, glass, polypropylene, agarose, gelatin, hydrogel,paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer,titanium, latex, sepharose, cellulose, nylon, silicone, or anycombination thereof. The barcoding particle can comprise a disruptablehydrogel particle.

In some embodiments, the barcoding particle is associated with anoptical moiety. The control oligonucleotide can be associated with anoptical moiety.

In some embodiments, the barcodes of the barcoding particle comprisemolecular label sequences selected from at least 1000, 10000, or moredifferent molecular label sequences. In some embodiments, the molecularlabel sequences of the barcodes comprise random sequences. In someembodiments, the barcoding particle comprises at least 10000 barcodes.

In some embodiments, barcoding the control oligonucleotides comprises:barcoding the control oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded controloligonucleotides. In some embodiments, barcoding the plurality ofcontrol oligonucleotides using the plurality of barcodes comprises:contacting the plurality of barcodes with control oligonucleotides ofthe plurality of control compositions to generate barcodes hybridized tothe control oligonucleotides; and extending the stochastic barcodeshybridized to the control oligonucleotides to generate the plurality ofbarcoded control oligonucleotides. Extending the barcodes can compriseextending the barcodes using a DNA polymerase, a reverse transcriptase,or a combination thereof. In some embodiments, the method comprisesamplifying the plurality of barcoded control oligonucleotides to producea plurality of amplicons. Amplifying the plurality of barcoded controloligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of the molecular label sequence andat least a portion of the control oligonucleotide. In some embodiments,obtaining the sequencing data comprises obtaining sequencing data of theplurality of amplicons. Obtaining the sequencing data can comprisesequencing the at least a portion of the molecular label sequence andthe at least a portion of the control oligonucleotide.

Disclosed herein include methods for sequencing control. In someembodiments, the method comprises: contacting one or more cells of aplurality of cells with a control composition of a plurality of controlcompositions, wherein a cell of the plurality of cells comprises aplurality of targets and a plurality of binding targets, wherein each ofthe plurality of control compositions comprises a cellular componentbinding reagent associated with a control oligonucleotide, wherein thecellular component binding reagent is capable of specifically binding toat least one of the plurality of binding targets, and wherein thecontrol oligonucleotide comprises a control barcode sequence and apseudo-target region comprising a sequence substantially complementaryto the target-binding region of at least one of the plurality ofbarcodes; barcoding the control oligonucleotides using a plurality ofbarcodes to create a plurality of barcoded control oligonucleotides,wherein each of the plurality of barcodes comprises a cell labelsequence, a molecular label sequence, and a target-binding region,wherein the molecular label sequences of at least two barcodes of theplurality of barcodes comprise different sequences, and wherein at leasttwo barcodes of the plurality of barcodes comprise an identical celllabel sequence; obtaining sequencing data of the plurality of barcodedcontrol oligonucleotides; determining at least one characteristic of theone or more cells using at least one characteristic of the plurality ofbarcoded control oligonucleotides in the sequencing data. In someembodiments, the pseudo-target region comprises a poly(dA) region.

In some embodiments, the control barcode sequence is at least 6nucleotides in length, 25-45 nucleotides in length, about 128nucleotides in length, at least 128 nucleotides in length, about 200-500nucleotides in length, or a combination thereof. The control particleoligonucleotide can be about 50 nucleotides in length, about 100nucleotides in length, about 200 nucleotides in length, at least 200nucleotides in length, less than about 200-300 nucleotides in length,about 500 nucleotides in length, or a combination thereof. The controlbarcode sequences of at least 2, 10, 100, 1000, or more of the pluralityof control particle oligonucleotides can be identical. At least 2, 10,100, 1000, or more of the plurality of control particle oligonucleotidescan comprise different control barcode sequences.

In some embodiments, determining the at least one characteristic of theone or more cells comprises: determining the number of cell labelsequences with distinct sequences associated with the plurality ofbarcoded control oligonucleotides in the sequencing data; anddetermining the number of the one or more cells using the number of celllabel sequences with distinct sequences associated with the plurality ofbarcoded control oligonucleotides. In some embodiments, the methodcomprises: determining single cell capture efficiency based the numberof the one or more cells determined. In some embodiments, the methodcomprises: determining single cell capture efficiency based on the ratioof the number of the one or more cells determined and the number of theplurality of cells.

In some embodiments, determining the at least one characteristic of theone or more cells can comprise: for each cell label in the sequencingdata, determining the number of molecular label sequences with distinctsequences associated with the cell label and the control barcodesequence; and determining the number of the one or more cells using thenumber of molecular label sequences with distinct sequences associatedwith the cell label and the control barcode sequence. Determining thenumber of molecular label sequences with distinct sequences associatedwith the cell label and the control barcode sequence comprises: for eachcell label in the sequencing data, determining the number of molecularlabel sequences with the highest number of distinct sequences associatedwith the cell label and the control barcode sequence. Determining thenumber of the one or more cells using the number of molecular labelsequences with distinct sequences associated with the cell label and thecontrol barcode sequence can comprise: generating a plot of the numberof molecular label sequences with the highest number of distinctsequences with the number of cell labels in the sequencing dataassociated with the number of molecular label sequences with the highestnumber of distinct sequences; and determining a cutoff in the plot asthe number of the one or more cells.

In some embodiments, the control oligonucleotide is not homologous togenomic sequences of any of the plurality of cells. The controloligonucleotide can be homologous to genomic sequences of a species. Thespecies can be a non-mammalian species. The non-mammalian species can bea phage species. The phage species can be T7 phage, a PhiX phage, or acombination thereof.

In some embodiments, the method comprises: releasing the controloligonucleotide from the cellular component binding reagent prior tobarcoding the control oligonucleotides. At least one of the plurality ofbinding targets can be expressed on a cell surface. At least one of theplurality of binding targets can comprise a cell-surface protein, a cellmarker, a B-cell receptor, a T-cell receptor, a major histocompatibilitycomplex, a tumor antigen, a receptor, an integrin, or any combinationthereof. The cellular component binding reagent can comprise a cellsurface binding reagent, an antibody, a tetramer, an aptamers, a proteinscaffold, an invasion, or a combination thereof.

In some embodiments, binding target of the cellular component bindingreagent is selected from a group comprising 10-100 different bindingtargets. Aa binding target of the cellular component binding reagent cancomprise a carbohydrate, a lipid, a protein, an extracellular protein, acell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, an intracellular protein, or any combinationthereof. The control oligonucleotide can be conjugated to the cellularcomponent binding reagent through a linker. The control oligonucleotidecan comprise the linker. The linker can comprise a chemical group. Thechemical group can be reversibly attached to the first cellularcomponent binding reagent. The chemical group can comprise a UVphotocleavable group, a streptavidin, a biotin, an amine, a disulfidelinkage, or any combination thereof.

In some embodiments, the cellular component binding reagent can beassociated with two or more control oligonucleotides with an identicalcontrol barcode sequence. The cellular component binding reagent can beassociated with two or more control oligonucleotides with differentidentical control barcode sequences. In some embodiments, a secondcellular component binding reagent of the plurality of controlcompositions is not associated with the control oligonucleotide. Thecellular component binding reagent and the second cellular componentbinding reagent can be identical.

In some embodiments, the barcode comprises a binding site for auniversal primer. In some embodiments, the target-binding regioncomprises a poly(dT) region.

In some embodiments, the plurality of barcodes is associated with abarcoding particle. At least one barcode of the plurality of barcodescan be immobilized on the barcoding particle. At least one barcode ofthe plurality of barcodes can be partially immobilized on the barcodingparticle. At least one barcode of the plurality of barcodes can beenclosed in the barcoding particle. At least one barcode of theplurality of barcodes can be partially enclosed in the barcodingparticle. The barcoding particle can be disruptable. The barcodingparticle can be a barcoding bead. In some embodiments, the barcodingbead comprises a Sepharose bead, a streptavidin bead, an agarose bead, amagnetic bead, a conjugated bead, a protein A conjugated bead, a proteinG conjugated bead, a protein A/G conjugated bead, a protein L conjugatedbead, an oligo(dT) conjugated bead, a silica bead, a silica-like bead,an anti-biotin microbead, an anti-fluorochrome microbead, or anycombination thereof. The barcoding particle can comprise a materialselected from the group consisting of polydimethylsiloxane (PDMS),polystyrene, glass, polypropylene, agarose, gelatin, hydrogel,paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer,titanium, latex, sepharose, cellulose, nylon, silicone, and acombination thereof. The barcoding particle can comprise a disruptablehydrogel particle. The barcoding particle can be associated with anoptical moiety.

In some embodiments, the control oligonucleotide can be associated withan optical moiety. In some embodiments, the barcodes of the barcodingparticle comprise molecular label sequences selected from at least 1000,10000, or more different molecular label sequences. In some embodiments,the molecular label sequences of the barcodes comprise random sequences.The barcoding particle can comprise at least 10000 barcodes.

In some embodiments, barcoding the control oligonucleotides comprises:barcoding the control oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded controloligonucleotides Barcoding the plurality of control oligonucleotidesusing the plurality of barcodes can comprise: contacting the pluralityof barcodes with control oligonucleotides of the plurality of controlcompositions to generate barcodes hybridized to the controloligonucleotides; and extending the stochastic barcodes hybridized tothe control oligonucleotides to generate the plurality of barcodedcontrol oligonucleotides. Extending the barcodes can comprise extendingthe barcodes using a DNA polymerase, a reverse transcriptase, or acombination thereof. In some embodiment, the method comprises amplifyingthe plurality of barcoded control oligonucleotides to produce aplurality of amplicons. Amplifying the plurality of barcoded controloligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of the molecular label sequence andat least a portion of the control oligonucleotide. Obtaining thesequencing data can comprise obtaining sequencing data of the pluralityof amplicons. Obtaining the sequencing data can comprise sequencing theat least a portion of the molecular label sequence and the at least aportion of the control oligonucleotide.

The methods for sequencing control can, in some embodiments, comprise:contacting one or more cells of a plurality of cells with a controlcomposition of a plurality of control compositions, wherein a cell ofthe plurality of cells comprises a plurality of targets and a pluralityof protein targets, wherein each of the plurality of controlcompositions comprises a protein binding reagent associated with acontrol oligonucleotide, wherein the protein binding reagent is capableof specifically binding to at least one of the plurality of proteintargets, and wherein the control oligonucleotide comprises a controlbarcode sequence and a pseudo-target region comprising a sequencesubstantially complementary to the target-binding region of at least oneof the plurality of barcodes; and determining at least onecharacteristic of the one or more cells using at least onecharacteristic of the plurality of control oligonucleotides. Thepseudo-target region can comprise a poly(dA) region.

In some embodiments, the control barcode sequence is at least 6nucleotides in length, 25-45 nucleotides in length, about 128nucleotides in length, at least 128 nucleotides in length, about 200-500nucleotides in length, or a combination thereof. The control particleoligonucleotide can be about 50 nucleotides in length, about 100nucleotides in length, about 200 nucleotides in length, at least 200nucleotides in length, less than about 200-300 nucleotides in length,about 500 nucleotides in length, or a combination thereof. The controlbarcode sequences of at least 2, 10, 100, 1000, or more of the pluralityof control particle oligonucleotides can be identical. At least 2, 10,100, 1000, or more of the plurality of control particle oligonucleotidescan comprise different control barcode sequences.

In some embodiments, determining the at least one characteristic of theone or more cells comprises: determining the number of cell labelsequences with distinct sequences associated with the plurality ofbarcoded control oligonucleotides in the sequencing data; anddetermining the number of the one or more cells using the number of celllabel sequences with distinct sequences associated with the plurality ofbarcoded control oligonucleotides. The method can comprise: determiningsingle cell capture efficiency based the number of the one or more cellsdetermined. The method can comprise: comprising determining single cellcapture efficiency based on the ratio of the number of the one or morecells determined and the number of the plurality of cells.

In some embodiments, determining the at least one characteristic of theone or more cells using the characteristics of the plurality of barcodedcontrol oligonucleotides in the sequencing data comprises: for each celllabel in the sequencing data, determining the number of molecular labelsequences with distinct sequences associated with the cell label and thecontrol barcode sequence; and determining the number of the one or morecells using the number of molecular label sequences with distinctsequences associated with the cell label and the control barcodesequence. Determining the number of molecular label sequences withdistinct sequences associated with the cell label and the controlbarcode sequence can comprise: for each cell label in the sequencingdata, determining the number of molecular label sequences with thehighest number of distinct sequences associated with the cell label andthe control barcode sequence. Determining the number of the one or morecells using the number of molecular label sequences with distinctsequences associated with the cell label and the control barcodesequence can comprise: generating a plot of the number of molecularlabel sequences with the highest number of distinct sequences with thenumber of cell labels in the sequencing data associated with the numberof molecular label sequences with the highest number of distinctsequences; and determining a cutoff in the plot as the number of the oneor more cells.

In some embodiments, the control oligonucleotide is not homologous togenomic sequences of any of the plurality of cells. The controloligonucleotide can be homologous to genomic sequences of a species. Thespecies can be a non-mammalian species. The non-mammalian species can bea phage species. The phage species can be T7 phage, a PhiX phage, or acombination thereof.

In some embodiments, the method comprises releasing the controloligonucleotide from the protein binding reagent prior to barcoding thecontrol oligonucleotides. In some embodiments, the method comprisesremoving unbound control compositions of the plurality of controlcompositions. Removing the unbound control compositions can comprisewashing the one or more cells of the plurality of cells with a washingbuffer. Removing the unbound sample indexing compositions can compriseselecting cells bound to at least one protein binding reagent of thecontrol composition using flow cytometry.

In some embodiments, at least one of the plurality of protein targets ison a cell surface. At least one of the plurality of protein targets cancomprise a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, or any combination thereof. The protein bindingreagent can comprise an antibody. The control oligonucleotide can beconjugated to the protein binding reagent through a linker. The controloligonucleotide can comprise the linker. The linker can comprise achemical group. The chemical group can be reversibly attached to thefirst protein binding reagent. The chemical group can comprise a UVphotocleavable group, a streptavidin, a biotin, an amine, a disulfidelinkage, or any combination thereof.

In some embodiments, the protein binding reagent is associated with twoor more control oligonucleotides with an identical control barcodesequence. The protein binding reagent can be associated with two or morecontrol oligonucleotides with different identical control barcodesequences. In some embodiments, a second protein binding reagent of theplurality of control compositions is not associated with the controloligonucleotide. The protein binding reagent and the second proteinbinding reagent can be identical.

In some embodiments, the barcode comprises a binding site for auniversal primer. The target-binding region can comprise a poly(dT)region. In some embodiments, the plurality of barcodes is associatedwith a barcoding particle. At least one barcode of the plurality ofbarcodes can be immobilized on the barcoding particle. At least onebarcode of the plurality of barcodes can be partially immobilized on thebarcoding particle. At least one barcode of the plurality of barcodes isenclosed in the barcoding particle. At least one barcode of theplurality of barcodes is partially enclosed in the barcoding particle.The barcoding particle can be disruptable. The barcoding particle can bea barcoding bead. The barcoding bead can comprise a Sepharose bead, astreptavidin bead, an agarose bead, a magnetic bead, a conjugated bead,a protein A conjugated bead, a protein G conjugated bead, a protein A/Gconjugated bead, a protein L conjugated bead, an oligo(dT) conjugatedbead, a silica bead, a silica-like bead, an anti-biotin microbead, ananti-fluorochrome microbead, or any combination thereof. The barcodingparticle can comprise a material of polydimethylsiloxane (PDMS),polystyrene, glass, polypropylene, agarose, gelatin, hydrogel,paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer,titanium, latex, sepharose, cellulose, nylon, silicone, or anycombination thereof. The barcoding particle can comprise a disruptablehydrogel particle.

In some embodiments, the barcoding particle is associated with anoptical moiety. The control oligonucleotide can be associated with anoptical moiety.

In some embodiments, the method comprises: barcoding the controloligonucleotides using a plurality of barcodes to create a plurality ofbarcoded control oligonucleotides, wherein each of the plurality ofbarcodes comprises a cell label sequence, a molecular label sequence,and a target-binding region, wherein the molecular label sequences of atleast two barcodes of the plurality of barcodes comprise differentsequences, and wherein at least two barcodes of the plurality ofbarcodes comprise an identical cell label sequence; and obtainingsequencing data of the plurality of barcoded control oligonucleotides;

In some embodiments, the barcodes of the barcoding particle comprisemolecular label sequences selected from at least 1000, 10000, or moredifferent molecular label sequences. In some embodiments, the molecularlabel sequences of the barcodes comprise random sequences. In someembodiments, the barcoding particle comprises at least 10000 barcodes.

In some embodiments, barcoding the control oligonucleotides comprises:barcoding the control oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded controloligonucleotides. In some embodiments, barcoding the plurality ofcontrol oligonucleotides using the plurality of barcodes comprises:contacting the plurality of barcodes with control oligonucleotides ofthe plurality of control compositions to generate barcodes hybridized tothe control oligonucleotides; and extending the stochastic barcodeshybridized to the control oligonucleotides to generate the plurality ofbarcoded control oligonucleotides. Extending the barcodes can compriseextending the barcodes using a DNA polymerase, a reverse transcriptase,or a combination thereof. In some embodiments, the method comprisesamplifying the plurality of barcoded control oligonucleotides to producea plurality of amplicons. Amplifying the plurality of barcoded controloligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of the molecular label sequence andat least a portion of the control oligonucleotide. In some embodiments,obtaining the sequencing data comprises obtaining sequencing data of theplurality of amplicons. Obtaining the sequencing data can comprisesequencing the at least a portion of the molecular label sequence andthe at least a portion of the control oligonucleotide.

Methods for cell identification can, in some embodiments, comprise:contacting a first plurality of cells and a second plurality of cellswith two sample indexing compositions respectively, wherein each of thefirst plurality of cells and each of the second plurality of cellscomprise one or more antigen targets, wherein each of the two sampleindexing compositions comprises an antigen binding reagent associatedwith a sample indexing oligonucleotide, wherein the antigen bindingreagent is capable of specifically binding to at least one of the one ormore antigen targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of the two sample indexing compositions comprise differentsequences; barcoding the sample indexing oligonucleotides using aplurality of barcodes to create a plurality of barcoded sample indexingoligonucleotides, wherein each of the plurality of barcodes comprises acell label sequence, a molecular label sequence, and a target-bindingregion, wherein the molecular label sequences of at least two barcodesof the plurality of barcodes comprise different sequences, and whereinat least two barcodes of the plurality of barcodes comprise an identicalcell label sequence; obtaining sequencing data of the plurality ofbarcoded sample indexing oligonucleotides; and identifying a cell labelsequence associated with two or more sample indexing sequences in thesequencing data obtained; and removing sequencing data associated withthe cell label sequence from the sequencing data obtained and/orexcluding the sequencing data associated with the cell label sequencefrom subsequent analysis. In some embodiments, the sample indexingoligonucleotide comprises a molecular label sequence, a binding site fora universal primer, or a combination thereof.

Disclosed herein also includes methods for multiplet identification. Amultiplet expression profile, or a multiplet, can be an expressionprofile comprising expression profiles of multiplet cells. Whendetermining expression profiles of single cells, n cells may beidentified as one cell and the expression profiles of the n cells may beidentified as the expression profile for one cell (referred to as amultiplet or n-plet expression profile). For example, when determiningexpression profiles of two cells using barcoding (e.g., stochasticbarcoding), the mRNA molecules of the two cells may be associated withbarcodes having the same cell label. As another example, two cells maybe associated with one particle (e.g., a bead). The particle can includebarcodes with the same cell label. After lysing the cells, the mRNAmolecules in the two cells can be associated with the barcodes of theparticle, thus the same cell label. Doublet expression profiles can skewthe interpretation of the expression profiles. Multiplets can bedifferent in different implementations. In some embodiments, theplurality of multiplets can include a doublet, a triplet, a quartet, aquintet, a sextet, a septet, an octet, a nonet, or any combinationthereof.

A multiplet can be any n-plet. In some embodiments, n is, or is about,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or arange between any two of these values. In some embodiments, n is atleast, or is at most, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20. A singlet can be an expression profile that isnot a multiplet expression profile.

The performance of using sample indexing oligonucleotides to indexsamples and identify multiplets can be comparable to the performance ofusing synthetic multiplet expression profiles to identify multiplets(described in U.S. application Ser. No. 15/926,977, filed on Mar. 20,2018, entitled “SYNTHETIC MULTIPLETS FOR MULTIPLETS DETERMINATION,” thecontent of which is incorporated herein in its entirety). In someembodiments, mutliplets can be identified using both sample indexingoligonucleotides and synthetic multiplet expression profiles.

In some embodiments, the methods of multiplet identification disclosedherein comprise: contacting a first plurality of cells and a secondplurality of cells with two sample indexing compositions respectively,wherein each of the first plurality of cells and each of the secondplurality of cells comprise one or more antigen targets, wherein each ofthe two sample indexing compositions comprises an antigen bindingreagent associated with a sample indexing oligonucleotide, wherein theantigen binding reagent is capable of specifically binding to at leastone of the one or more antigen targets, wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of the two sample indexing compositions comprisedifferent sequences; barcoding the sample indexing oligonucleotidesusing a plurality of barcodes to create a plurality of barcoded sampleindexing oligonucleotides, wherein each of the plurality of barcodescomprises a cell label sequence, a molecular label sequence, and atarget-binding region, wherein the molecular label sequences of at leasttwo barcodes of the plurality of barcodes comprise different sequences,and wherein at least two barcodes of the plurality of barcodes comprisean identical cell label sequence; obtaining sequencing data of theplurality of barcoded sample indexing oligonucleotides; and identifyingone or more multiplet cell label sequences that is each associated withtwo or more sample indexing sequences in the sequencing data obtained.In some embodiments, the method comprises: removing the sequencing dataassociated with the one or more multiplet cell label sequences from thesequencing data obtained and/or excluding the sequencing data associatedwith the one or more multiplet cell label sequences from subsequentanalysis. In some embodiments, the sample indexing oligonucleotidecomprises a molecular label sequence, a binding site for a universalprimer, or a combination thereof.

In some embodiments, contacting the first plurality of cells and thesecond plurality of cells with the two sample indexing compositionsrespectively comprises: contacting the first plurality of cells with afirst sample indexing compositions of the two sample indexingcompositions; and contacting the first plurality of cells with a secondsample indexing compositions of the two sample indexing compositions.

In some embodiments, the sample indexing sequence is at least 6nucleotides in length, 25-60 nucleotides in length (e.g., 45 nucleotidesin length), about 128 nucleotides in length, at least 128 nucleotides inlength, about 200-500 nucleotides in length, or a combination thereof.The sample indexing oligonucleotide can be about 50 nucleotides inlength, about 100 nucleotides in length, about 200 nucleotides inlength, at least 200 nucleotides in length, less than about 200-300nucleotides in length, about 500 nucleotides in length, or a combinationthereof. In some embodiments, sample indexing sequences of at least 10,100, 1000, or more sample indexing compositions of the plurality ofsample indexing compositions comprise different sequences.

In some embodiments, the antigen binding reagent comprises an antibody,a tetramer, an aptamers, a protein scaffold, or a combination thereof.The sample indexing oligonucleotide can be conjugated to the antigenbinding reagent through a linker. The oligonucleotide can comprise thelinker. The linker can comprise a chemical group. The chemical group canbe reversibly or irreversibly attached to the antigen binding reagent.The chemical group can comprise a UV photocleavable group, a disulfidebond, a streptavidin, a biotin, an amine, a disulfide linkage or anycombination thereof.

In some embodiments, at least one of the first plurality of cells andthe second plurality of cells comprises single cells. The at least oneof the one or more antigen targets can be on a cell surface.

In some embodiments, the method comprises: removing unbound sampleindexing compositions of the two sample indexing compositions. Removingthe unbound sample indexing compositions can comprise washing cells ofthe first plurality of cells and the second plurality of cells with awashing buffer. Removing the unbound sample indexing compositions cancomprise selecting cells bound to at least one antigen binding reagentof the two sample indexing compositions using flow cytometry. In someembodiments, the method comprises: lysing one or more cells of the firstplurality of cells and the second plurality of cells.

In some embodiments, the sample indexing oligonucleotide is configuredto be detachable or non-detachable from the antigen binding reagent. Themethod can comprise detaching the sample indexing oligonucleotide fromthe antigen binding reagent. Detaching the sample indexingoligonucleotide can comprise detaching the sample indexingoligonucleotide from the antigen binding reagent by UV photocleaving,chemical treatment (e.g., using reducing reagent, such asdithiothreitol), heating, enzyme treatment, or any combination thereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of any of the one or more cells. Thecontrol barcode sequence may be not homologous to genomic sequences of aspecies. The species can be a non-mammalian species. The non-mammalianspecies can be a phage species. The phage species is T7 phage, a PhiXphage, or a combination thereof.

In some embodiments, the first plurality of cells and the secondplurality of cells comprise a tumor cells, a mammalian cell, a bacterialcell, a viral cell, a yeast cell, a fungal cell, or any combinationthereof. The sample indexing oligonucleotide can comprise a sequencecomplementary to a capture sequence of at least one barcode of theplurality of barcodes. The barcode can comprise a target-binding regionwhich comprises the capture sequence. The target-binding region cancomprise a poly(dT) region. The sequence of the sample indexingoligonucleotide complementary to the capture sequence of the barcode cancomprise a poly(dA) region.

In some embodiments, the antigen target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. The antigen target can be, or comprise, a cell-surface protein,a cell marker, a B-cell receptor, a T-cell receptor, a majorhistocompatibility complex, a tumor antigen, a receptor, an integrin, orany combination thereof. The antigen target can be, or comprise, alipid, a carbohydrate, or any combination thereof. The antigen targetcan be selected from a group comprising 10-100 different antigentargets.

In some embodiments, the antigen binding reagent is associated with twoor more sample indexing oligonucleotides with an identical sequence. Theantigen binding reagent can be associated with two or more sampleindexing oligonucleotides with different sample indexing sequences. Thesample indexing composition of the plurality of sample indexingcompositions can comprise a second antigen binding reagent notconjugated with the sample indexing oligonucleotide. The antigen bindingreagent and the second antigen binding reagent can be identical.

In some embodiments, a barcode of the plurality of barcodes comprises atarget-binding region and a molecular label sequence, and molecularlabel sequences of at least two barcodes of the plurality of barcodescomprise different molecule label sequences. The barcode can comprise acell label sequence, a binding site for a universal primer, or anycombination thereof. The target-binding region can comprise a poly(dT)region.

In some embodiments, the plurality of barcodes can be associated with aparticle. At least one barcode of the plurality of barcodes can beimmobilized on the particle. At least one barcode of the plurality ofbarcodes can be partially immobilized on the particle. At least onebarcode of the plurality of barcodes can be enclosed in the particle. Atleast one barcode of the plurality of barcodes can be partially enclosedin the particle. The particle can be disruptable. The particle can be abead. The bead can be, or comprise, a Sepharose bead, a streptavidinbead, an agarose bead, a magnetic bead, a conjugated bead, a protein Aconjugated bead, a protein G conjugated bead, a protein A/G conjugatedbead, a protein L conjugated bead, an oligo(dT) conjugated bead, asilica bead, a silica-like bead, an anti-biotin microbead, ananti-fluorochrome microbead, or any combination thereof. The particlecan comprise a material selected from the group consisting ofpolydimethylsiloxane/(PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone,and any combination thereof. The particle can comprise a disruptablehydrogel particle.

In some embodiments, the antigen binding reagent is associated with adetectable moiety. In some embodiments, the particle is associated witha detectable moiety. The sample indexing oligonucleotide is associatedwith an optical moiety. In some embodiments, the barcodes of theparticle can comprise molecular label sequences selected from at least1000, 10000, or more different molecular label sequences. The molecularlabel sequences of the barcodes can comprise random sequences. Theparticle can comprise at least 10000 barcodes.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises: amplifying the plurality ofbarcoded sample indexing oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded sample indexingoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of the molecular label sequence andat least a portion of the sample indexing oligonucleotide. In someembodiments, obtaining the sequencing data of the plurality of barcodedsample indexing oligonucleotides can comprise obtaining sequencing dataof the plurality of amplicons. Obtaining the sequencing data comprisessequencing at least a portion of the molecular label sequence and atleast a portion of the sample indexing oligonucleotide. In someembodiments, identifying the sample origin of the at least one cellcomprises identifying sample origin of the plurality of barcoded targetsbased on the sample indexing sequence of the at least one barcodedsample indexing oligonucleotide.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes to create the plurality of barcodedsample indexing oligonucleotides comprises stochastically barcoding thesample indexing oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label sequence, and wherein at least two barcodes ofthe plurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can comprise: contacting copies of thetargets with target-binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets.

In some embodiments, the method comprises: prior to obtaining thesequencing data of the plurality of barcoded targets, amplifying thebarcoded targets to create a plurality of amplified barcoded targets.Amplifying the barcoded targets to generate the plurality of amplifiedbarcoded targets can comprise: amplifying the barcoded targets bypolymerase chain reaction (PCR). Barcoding the plurality of targets ofthe cell using the plurality of barcodes to create the plurality ofbarcoded targets can comprise stochastically barcoding the plurality oftargets of the cell using a plurality of stochastic barcodes to create aplurality of stochastically barcoded targets.

Methods for cell identification can, in some embodiments, comprise:contacting a first plurality of cells and a second plurality of cellswith two sample indexing compositions respectively, wherein each of thefirst plurality of cells and each of the second plurality of cellscomprise one or more cellular component targets, wherein each of the twosample indexing compositions comprises a cellular component bindingreagent associated with a sample indexing oligonucleotide, wherein thecellular component binding reagent is capable of specifically binding toat least one of the one or more cellular component targets, wherein thesample indexing oligonucleotide comprises a sample indexing sequence,and wherein sample indexing sequences of the two sample indexingcompositions of the plurality of sample indexing compositions comprisedifferent sequences; barcoding the sample indexing oligonucleotidesusing a plurality of barcodes to create a plurality of barcoded sampleindexing oligonucleotides, wherein each of the plurality of barcodescomprises a cell label sequence, a molecular label sequence, and atarget-binding region, wherein the molecular label sequences of at leasttwo barcodes of the plurality of barcodes comprise different sequences,and wherein at least two barcodes of the plurality of barcodes comprisean identical cell label sequence; obtaining sequencing data of theplurality of barcoded sample indexing oligonucleotides; identifying oneor more cell label sequences that is each associated with two or moresample indexing sequences in the sequencing data obtained; and removingthe sequencing data associated with the one or more cell label sequencesthat is each associated with two or more sample indexing sequences fromthe sequencing data obtained and/or excluding the sequencing dataassociated with the one or more cell label sequences that is eachassociated with two or more sample indexing sequences from subsequentanalysis. In some embodiments, the sample indexing oligonucleotidecomprises a molecular label sequence, a binding site for a universalprimer, or a combination thereof.

Disclosed herein includes methods for multiplet identification. In someembodiments, the method comprises: contacting a first plurality of cellsand a second plurality of cells with two sample indexing compositionsrespectively, wherein each of the first plurality of cells and each ofthe second plurality of cells comprise one or more cellular componenttargets, wherein each of the two sample indexing compositions comprisesa cellular component binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of the two sample indexing compositions of the plurality ofsample indexing compositions comprise different sequences; barcoding thesample indexing oligonucleotides using a plurality of barcodes to createa plurality of barcoded sample indexing oligonucleotides, wherein eachof the plurality of barcodes comprises a cell label sequence, amolecular label sequence, and a target-binding region, wherein themolecular label sequences of at least two barcodes of the plurality ofbarcodes comprise different sequences, and wherein at least two barcodesof the plurality of barcodes comprise an identical cell label sequence;obtaining sequencing data of the plurality of barcoded sample indexingoligonucleotides; identifying one or more multiplet cell label sequencesthat is each associated with two or more sample indexing sequences inthe sequencing data obtained. In some embodiments, the method comprises:removing the sequencing data associated with the one or more multipletcell label sequences from the sequencing data obtained and/or excludingthe sequencing data associated with the one or more multiplet cell labelsequences from subsequent analysis. In some embodiments, the sampleindexing oligonucleotide comprises a molecular label sequence, a bindingsite for a universal primer, or a combination thereof.

In some embodiments, contacting the first plurality of cells and thesecond plurality of cells with the two sample indexing compositionsrespectively comprises: contacting the first plurality of cells with afirst sample indexing compositions of the two sample indexingcompositions; and contacting the first plurality of cells with a secondsample indexing compositions of the two sample indexing compositions.

In some embodiments, the sample indexing sequence is at least 6nucleotides in length, 25-60 nucleotides in length (e.g., 45 nucleotidesin length), about 128 nucleotides in length, at least 128 nucleotides inlength, about 200-500 nucleotides in length, or a combination thereof.The sample indexing oligonucleotide can be about 50 nucleotides inlength, about 100 nucleotides in length, about 200 nucleotides inlength, at least 200 nucleotides in length, less than about 200-300nucleotides in length, about 500 nucleotides in length, or a combinationthereof. In some embodiments, sample indexing sequences of at least 10,100, 1000, or more sample indexing compositions of the plurality ofsample indexing compositions comprise different sequences.

In some embodiments, the cellular component binding reagent comprises anantibody, a tetramer, an aptamers, a protein scaffold, or a combinationthereof. The sample indexing oligonucleotide can be conjugated to thecellular component binding reagent through a linker. The oligonucleotidecan comprise the linker. The linker can comprise a chemical group. Thechemical group can be reversibly or irreversibly attached to thecellular component binding reagent. The chemical group can comprise a UVphotocleavable group, a disulfide bond, a streptavidin, a biotin, anamine, a disulfide linkage or any combination thereof.

In some embodiments, at least one of the first plurality of cells andthe second plurality of cells comprises a single cell. The at least oneof the one or more cellular component targets can be on a cell surface.

In some embodiments, the method comprises: removing unbound sampleindexing compositions of the two sample indexing compositions. Removingthe unbound sample indexing compositions can comprise washing cells ofthe first plurality of cells and the second plurality of cells with awashing buffer. Removing the unbound sample indexing compositions cancomprise selecting cells bound to at least one cellular componentbinding reagent of the two sample indexing compositions using flowcytometry. In some embodiments, the method comprises: lysing one or morecells of the first plurality of cells and the second plurality of cells.

In some embodiments, the sample indexing oligonucleotide is configuredto be detachable or non-detachable from the cellular component bindingreagent. The method can comprise detaching the sample indexingoligonucleotide from the cellular component binding reagent. Detachingthe sample indexing oligonucleotide can comprise detaching the sampleindexing oligonucleotide from the cellular component binding reagent byUV photocleaving, chemical treatment (e.g., using reducing reagent, suchas dithiothreitol), heating, enzyme treatment, or any combinationthereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of any of the one or more cells. Thecontrol barcode sequence may be not homologous to genomic sequences of aspecies. The species can be a non-mammalian species. The non-mammalianspecies can be a phage species. The phage species is T7 phage, a PhiXphage, or a combination thereof.

In some embodiments, the first plurality of cells and the secondplurality of cells comprise a tumor cell, a mammalian cell, a bacterialcell, a viral cell, a yeast cell, a fungal cell, or any combinationthereof. The sample indexing oligonucleotide can comprise a sequencecomplementary to a capture sequence of at least one barcode of theplurality of barcodes. The barcode can comprise a target-binding regionwhich comprises the capture sequence. The target-binding region cancomprise a poly(dT) region. The sequence of the sample indexingoligonucleotide complementary to the capture sequence of the barcode cancomprise a poly(dA) region.

In some embodiments, the antigen target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. The antigen target can be, or comprise, a cell-surface protein,a cell marker, a B-cell receptor, a T-cell receptor, a majorhistocompatibility complex, a tumor antigen, a receptor, an integrin, orany combination thereof. The antigen target can be, or comprise, alipid, a carbohydrate, or any combination thereof. The antigen targetcan be selected from a group comprising 10-100 different antigentargets.

In some embodiments, the cellular component binding reagent isassociated with two or more sample indexing oligonucleotides with anidentical sequence. The cellular component binding reagent can beassociated with two or more sample indexing oligonucleotides withdifferent sample indexing sequences. The sample indexing composition ofthe plurality of sample indexing compositions can comprise a secondcellular component binding reagent not conjugated with the sampleindexing oligonucleotide. The cellular component binding reagent and thesecond cellular component binding reagent can be identical.

In some embodiments, a barcode of the plurality of barcodes comprises atarget-binding region and a molecular label sequence, and molecularlabel sequences of at least two barcodes of the plurality of barcodescomprise different molecule label sequences. The barcode can comprise acell label sequence, a binding site for a universal primer, or anycombination thereof. The target-binding region can comprise a poly(dT)region.

In some embodiments, the plurality of barcodes can be associated with aparticle. At least one barcode of the plurality of barcodes can beimmobilized on the particle. At least one barcode of the plurality ofbarcodes can be partially immobilized on the particle. At least onebarcode of the plurality of barcodes can be enclosed in the particle. Atleast one barcode of the plurality of barcodes can be partially enclosedin the particle. The particle can be disruptable. The particle can be abead. The bead can be, or comprise, a Sepharose bead, a streptavidinbead, an agarose bead, a magnetic bead, a conjugated bead, a protein Aconjugated bead, a protein G conjugated bead, a protein A/G conjugatedbead, a protein L conjugated bead, an oligo(dT) conjugated bead, asilica bead, a silica-like bead, an anti-biotin microbead, ananti-fluorochrome microbead, or any combination thereof. The particlecan comprise a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone,and any combination thereof. The particle can comprise a disruptablehydrogel particle.

In some embodiments, the cellular component binding reagent isassociated with a detectable moiety. In some embodiments, the particleis associated with a detectable moiety. The sample indexingoligonucleotide is associated with an optical moiety.

In some embodiments, the barcodes of the particle can comprise molecularlabel sequences selected from at least 1000, 10000, or more differentmolecular label sequences. The molecular label sequences of the barcodescan comprise random sequences. The particle can comprise at least 10000barcodes.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises: amplifying the plurality ofbarcoded sample indexing oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded sample indexingoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of the molecular label sequence andat least a portion of the sample indexing oligonucleotide. In someembodiments, obtaining the sequencing data of the plurality of barcodedsample indexing oligonucleotides can comprise obtaining sequencing dataof the plurality of amplicons. Obtaining the sequencing data comprisessequencing at least a portion of the molecular label sequence and atleast a portion of the sample indexing oligonucleotide. In someembodiments, identifying the sample origin of the at least one cellcomprises identifying sample origin of the plurality of barcoded targetsbased on the sample indexing sequence of the at least one barcodedsample indexing oligonucleotide.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes to create the plurality of barcodedsample indexing oligonucleotides comprises stochastically barcoding thesample indexing oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label sequence, and wherein at least two barcodes ofthe plurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can comprise: contacting copies of thetargets with target-binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets.

In some embodiments, the method comprises: prior to obtaining thesequencing data of the plurality of barcoded targets, amplifying thebarcoded targets to create a plurality of amplified barcoded targets.Amplifying the barcoded targets to generate the plurality of amplifiedbarcoded targets can comprise: amplifying the barcoded targets bypolymerase chain reaction (PCR). Barcoding the plurality of targets ofthe cell using the plurality of barcodes to create the plurality ofbarcoded targets can comprise stochastically barcoding the plurality oftargets of the cell using a plurality of stochastic barcodes to create aplurality of stochastically barcoded targets.

Disclosed herein includes methods for cell identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a first plurality of cells and a second plurality of cells witha sample indexing composition of a plurality of two sample indexingcompositions respectively, wherein each of the first plurality of cellsand each of the second plurality of cells comprises one or more antigentargets, wherein each of the two sample indexing compositions comprisesan antigen binding reagent associated with a sample indexingoligonucleotide, wherein the antigen binding reagent is capable ofspecifically binding to at least one of the one or more antigen targets,wherein the sample indexing oligonucleotide comprises a sample indexingsequence, and wherein sample indexing sequences of the two sampleindexing compositions comprise different sequences; and identifying oneor more cells that is each associated with two or more sample indexingsequences. In some embodiments, the sample indexing oligonucleotidecomprises a molecular label sequence, a binding site for a universalprimer, or a combination thereof.

Disclosed herein include methods for multiplet identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a first plurality of cells and a second plurality of cells witha sample indexing composition of a plurality of two sample indexingcompositions respectively, wherein each of the first plurality of cellsand each of the second plurality of cells comprises one or more antigentargets, wherein each of the two sample indexing compositions comprisesan antigen binding reagent associated with a sample indexingoligonucleotide, wherein the antigen binding reagent is capable ofspecifically binding to at least one of the one or more antigen targets,wherein the sample indexing oligonucleotide comprises a sample indexingsequence, and wherein sample indexing sequences of the two sampleindexing compositions comprise different sequences; and identifying oneor more cells that is each associated with two or more sample indexingsequences as multiplet cells.

In some embodiments, identifying the cells that is each associated withtwo or more sample indexing sequences comprises: barcoding the sampleindexing oligonucleotides using a plurality of barcodes to create aplurality of barcoded sample indexing oligonucleotides, wherein each ofthe plurality of barcodes comprises a cell label sequence, a molecularlabel sequence, and a target-binding region, wherein the molecular labelsequences of at least two barcodes of the plurality of barcodes comprisedifferent sequences, and wherein at least two barcodes of the pluralityof barcodes comprise an identical cell label sequence; obtainingsequencing data of the plurality of barcoded sample indexingoligonucleotides; and identifying one or more cell label sequences thatis each associated with two or more sample indexing sequences in thesequencing data obtained. The method can comprise removing thesequencing data associated with the one or more cell label sequencesthat is each associated with two or more sample indexing sequences fromthe sequencing data obtained and/or excluding the sequencing dataassociated with the one or more cell label sequences that is eachassociated with the two or more sample indexing sequences fromsubsequent analysis.

In some embodiments, contacting the first plurality of cells and thesecond plurality of cells with the two sample indexing compositionsrespectively comprises: contacting the first plurality of cells with afirst sample indexing compositions of the two sample indexingcompositions; and contacting the first plurality of cells with a secondsample indexing compositions of the two sample indexing compositions.

In some embodiments, the sample indexing sequence is at least 6nucleotides in length, 25-60 nucleotides in length (e.g., 45 nucleotidesin length), about 128 nucleotides in length, at least 128 nucleotides inlength, about 200-500 nucleotides in length, or a combination thereof.The sample indexing oligonucleotide can be about 50 nucleotides inlength, about 100 nucleotides in length, about 200 nucleotides inlength, at least 200 nucleotides in length, less than about 200-300nucleotides in length, about 500 nucleotides in length, or a combinationthereof. In some embodiments, sample indexing sequences of at least 10,100, 1000, or more sample indexing compositions of the plurality ofsample indexing compositions comprise different sequences.

In some embodiments, the antigen binding reagent comprises an antibody,a tetramer, an aptamers, a protein scaffold, or a combination thereof.The sample indexing oligonucleotide can be conjugated to the antigenbinding reagent through a linker. The oligonucleotide can comprise thelinker. The linker can comprise a chemical group. The chemical group canbe reversibly or irreversibly attached to the antigen binding reagent.The chemical group can comprise a UV photocleavable group, a disulfidebond, a streptavidin, a biotin, an amine, a disulfide linkage or anycombination thereof.

In some embodiments, at least one of the first plurality of cells andthe second plurality of cells comprises single cells. The at least oneof the one or more antigen targets can be on a cell surface. In someembodiments, the method comprises: removing unbound sample indexingcompositions of the two sample indexing compositions. Removing theunbound sample indexing compositions can comprise washing cells of thefirst plurality of cells and the second plurality of cells with awashing buffer. Removing the unbound sample indexing compositions cancomprise selecting cells bound to at least one antigen binding reagentof the two sample indexing compositions using flow cytometry. In someembodiments, the method comprises: lysing one or more cells of the firstplurality of cells and the second plurality of cells.

In some embodiments, the sample indexing oligonucleotide is configuredto be detachable or non-detachable from the antigen binding reagent. Themethod can comprise detaching the sample indexing oligonucleotide fromthe antigen binding reagent. Detaching the sample indexingoligonucleotide can comprise detaching the sample indexingoligonucleotide from the antigen binding reagent by UV photocleaving,chemical treatment (e.g., using reducing reagent, such asdithiothreitol), heating, enzyme treatment, or any combination thereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of any of the one or more cells. Thecontrol barcode sequence may be not homologous to genomic sequences of aspecies. The species can be a non-mammalian species. The non-mammalianspecies can be a phage species. The phage species is T7 phage, a PhiXphage, or a combination thereof.

In some embodiments, the first plurality of cells and the secondplurality of cells comprise a tumor cells, a mammalian cell, a bacterialcell, a viral cell, a yeast cell, a fungal cell, or any combinationthereof. The sample indexing oligonucleotide can comprise a sequencecomplementary to a capture sequence of at least one barcode of theplurality of barcodes. The barcode can comprise a target-binding regionwhich comprises the capture sequence. The target-binding region cancomprise a poly(dT) region. The sequence of the sample indexingoligonucleotide complementary to the capture sequence of the barcode cancomprise a poly(dA) region.

In some embodiments, the antigen target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. The antigen target can be, or comprise, a cell-surface protein,a cell marker, a B-cell receptor, a T-cell receptor, a majorhistocompatibility complex, a tumor antigen, a receptor, an integrin, orany combination thereof. The antigen target can be, or comprise, alipid, a carbohydrate, or any combination thereof. The antigen targetcan be selected from a group comprising 10-100 different antigentargets.

In some embodiments, the antigen binding reagent is associated with twoor more sample indexing oligonucleotides with an identical sequence. Theantigen binding reagent can be associated with two or more sampleindexing oligonucleotides with different sample indexing sequences. Thesample indexing composition of the plurality of sample indexingcompositions can comprise a second antigen binding reagent notconjugated with the sample indexing oligonucleotide. The antigen bindingreagent and the second antigen binding reagent can be identical.

In some embodiments, a barcode of the plurality of barcodes comprises atarget-binding region and a molecular label sequence, and molecularlabel sequences of at least two barcodes of the plurality of barcodescomprise different molecule label sequences. The barcode can comprise acell label sequence, a binding site for a universal primer, or anycombination thereof. The target-binding region can comprise a poly(dT)region.

In some embodiments, the plurality of barcodes can be associated with aparticle. At least one barcode of the plurality of barcodes can beimmobilized on the particle. At least one barcode of the plurality ofbarcodes can be partially immobilized on the particle. At least onebarcode of the plurality of barcodes can be enclosed in the particle. Atleast one barcode of the plurality of barcodes can be partially enclosedin the particle. The particle can be disruptable. The particle can be abead. The bead can be, or comprise, a Sepharose bead, a streptavidinbead, an agarose bead, a magnetic bead, a conjugated bead, a protein Aconjugated bead, a protein G conjugated bead, a protein A/G conjugatedbead, a protein L conjugated bead, an oligo(dT) conjugated bead, asilica bead, a silica-like bead, an anti-biotin microbead, ananti-fluorochrome microbead, or any combination thereof. The particlecan comprise a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone,and any combination thereof. The particle can comprise a disruptablehydrogel particle.

In some embodiments, the antigen binding reagent is associated with adetectable moiety. In some embodiments, the particle is associated witha detectable moiety. The sample indexing oligonucleotide is associatedwith an optical moiety.

In some embodiments, the barcodes of the particle can comprise molecularlabel sequences selected from at least 1000, 10000, or more differentmolecular label sequences. The molecular label sequences of the barcodescan comprise random sequences. The particle can comprise at least 10000barcodes.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises: amplifying the plurality ofbarcoded sample indexing oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded sample indexingoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of the molecular label sequence andat least a portion of the sample indexing oligonucleotide. In someembodiments, obtaining the sequencing data of the plurality of barcodedsample indexing oligonucleotides can comprise obtaining sequencing dataof the plurality of amplicons. Obtaining the sequencing data comprisessequencing at least a portion of the molecular label sequence and atleast a portion of the sample indexing oligonucleotide. In someembodiments, identifying the sample origin of the at least one cellcomprises identifying sample origin of the plurality of barcoded targetsbased on the sample indexing sequence of the at least one barcodedsample indexing oligonucleotide.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes to create the plurality of barcodedsample indexing oligonucleotides comprises stochastically barcoding thesample indexing oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label sequence, and wherein at least two barcodes ofthe plurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can comprise: contacting copies of thetargets with target-binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets.

In some embodiments, the method comprises: prior to obtaining thesequencing data of the plurality of barcoded targets, amplifying thebarcoded targets to create a plurality of amplified barcoded targets.Amplifying the barcoded targets to generate the plurality of amplifiedbarcoded targets can comprise: amplifying the barcoded targets bypolymerase chain reaction (PCR). Barcoding the plurality of targets ofthe cell using the plurality of barcodes to create the plurality ofbarcoded targets can comprise stochastically barcoding the plurality oftargets of the cell using a plurality of stochastic barcodes to create aplurality of stochastically barcoded targets.

Disclosed herein include systems, methods, and kits for determininginteractions between cellular component targets, for exampleinteractions between proteins. In some embodiments, the methodcomprises: contacting a cell with a first pair of interactiondetermination compositions, wherein the cell comprises a first proteintarget and a second protein target, wherein each of the first pair ofinteraction determination compositions comprises a protein bindingreagent associated with an interaction determination oligonucleotide,wherein the protein binding reagent of one of the first pair ofinteraction determination compositions is capable of specificallybinding to the first protein target and the protein binding reagent ofthe other of the first pair of interaction determination compositions iscapable of specifically binding to the second protein target, andwherein the interaction determination oligonucleotide comprises aninteraction determination sequence and a bridge oligonucleotidehybridization region, and wherein the interaction determinationsequences of the first pair of interaction determination compositionscomprise different sequences; ligating the interaction determinationoligonucleotides of the first pair of interaction determinationcompositions using a bridge oligonucleotide to generate a ligatedinteraction determination oligonucleotide, wherein the bridgeoligonucleotide comprises two hybridization regions capable ofspecifically binding to the bridge oligonucleotide hybridization regionsof the first pair of interaction determination compositions; barcodingthe ligated interaction determination oligonucleotide using a pluralityof barcodes to create a plurality of barcoded interaction determinationoligonucleotides, wherein each of the plurality of barcodes comprises abarcode sequence and a capture sequence; obtaining sequencing data ofthe plurality of barcoded interaction determination oligonucleotides;and determining an interaction between the first and second proteintargets based on the association of the interaction determinationsequences of the first pair of interaction determination compositions inthe obtained sequencing data.

Disclosed herein include systems, methods, and kits for determininginteractions between cellular component targets. In some embodiments,the method comprises: contacting a cell with a first pair of interactiondetermination compositions, wherein the cell comprises a first cellularcomponent target and a second cellular component target, wherein each ofthe first pair of interaction determination compositions comprises acellular component binding reagent associated with an interactiondetermination oligonucleotide, wherein the cellular component bindingreagent of one of the first pair of interaction determinationcompositions is capable of specifically binding to the first cellularcomponent target and the cellular component binding reagent of the otherof the first pair of interaction determination compositions is capableof specifically binding to the second cellular component target, andwherein the interaction determination oligonucleotide comprises aninteraction determination sequence and a bridge oligonucleotidehybridization region, and wherein the interaction determinationsequences of the first pair of interaction determination compositionscomprise different sequences; ligating the interaction determinationoligonucleotides of the first pair of interaction determinationcompositions using a bridge oligonucleotide to generate a ligatedinteraction determination oligonucleotide, wherein the bridgeoligonucleotide comprises two hybridization regions capable ofspecifically binding to the bridge oligonucleotide hybridization regionsof the first pair of interaction determination compositions; barcodingthe ligated interaction determination oligonucleotide using a pluralityof barcodes to create a plurality of barcoded interaction determinationoligonucleotides, wherein each of the plurality of barcodes comprises abarcode sequence and a capture sequence; obtaining sequencing data ofthe plurality of barcoded interaction determination oligonucleotides;and determining an interaction between the first and second cellularcomponent targets based on the association of the interactiondetermination sequences of the first pair of interaction determinationcompositions in the obtained sequencing data. At least one of the twocellular component binding reagent can comprise a protein bindingreagent. The protein binding reagent can be associated with one of thetwo interaction determination oligonucleotides. The one or more cellularcomponent targets can comprise at least one protein target.

In some embodiments, contacting the cell with the first pair ofinteraction determination compositions comprises: contacting the cellwith each of the first pair of interaction determination compositionssequentially or simultaneously. The first protein target can be the sameas the second protein target, or the first protein target can bedifferent from the second protein target.

The length of the interaction determination sequence can vary. Forexample, the interaction determination sequence can be 2 nucleotides toabout 1000 nucleotides in length. In some embodiments, the interactiondetermination sequence can be at least 6 nucleotides in length, 25-60nucleotides in length, about 45 nucleotides in length, about 50nucleotides in length, about 100 nucleotides in length, about 128nucleotides in length, at least 128 nucleotides in length, about 200nucleotides in length, at least 200 nucleotides in length, about 200-300nucleotides in length, about 200-500 nucleotides in length, about 500nucleotides in length, or any combination thereof.

In some embodiments, the method comprises: contacting the cell with asecond pair of interaction determination compositions, wherein the cellcomprises a third protein target and a fourth protein target, whereineach of the second pair of interaction determination compositionscomprises a protein binding reagent associated with an interactiondetermination oligonucleotide, wherein the protein binding reagent ofone of the second pair of interaction determination compositions iscapable of specifically binding to the third protein target and theprotein binding reagent of the other of the second pair of interactiondetermination compositions is capable of specifically binding to thefourth protein target. At least one of the third and fourth proteintargets can be different from one of the first and second proteintargets. In some embodiments, at least one of the third and fourthprotein targets and at least one of the first and second protein targetscan be identical.

In some embodiments, the method comprises: contacting the cell withthree or more pairs of interaction determination compositions. Theinteraction determination sequences of at least 10 interactiondetermination compositions of the plurality of pairs of interactiondetermination compositions can comprise different sequences. Theinteraction determination sequences of at least 100 interactiondetermination compositions of the plurality of pairs of interactiondetermination compositions can comprise different sequences. Theinteraction determination sequences of at least 1000 interactiondetermination compositions of the plurality of pairs of interactiondetermination compositions can comprise different sequences.

In some embodiments, the bridge oligonucleotide hybridization regions ofthe first pair of interaction determination compositions comprisedifferent sequences. At least one of the bridge oligonucleotidehybridization regions can be complementary to at least one of the twohybridization regions of the bridge oligonucleotide.

In some embodiments, ligating the interaction determinationoligonucleotides of the first pair of interaction determinationcompositions using the bridge oligonucleotide comprises: hybridizing afirst hybridization regions of the bridge oligonucleotide with a firstbridge oligonucleotide hybridization region of the bridgeoligonucleotide hybridization regions of the interaction determinationoligonucleotides; hybridizing a second hybridization region of thebridge oligonucleotide with a second bridge oligonucleotidehybridization region of the bridge oligonucleotide hybridization regionsof the interaction determination oligonucleotides; and ligating theinteraction determination oligonucleotides hybridized to the bridgeoligonucleotide to generate a ligated interaction determinationoligonucleotide.

In some embodiments, the protein binding reagent comprises an antibody,a tetramer, an aptamers, a protein scaffold, an integrin, or acombination thereof. The interaction determination oligonucleotide canbe conjugated to the protein binding reagent through a linker. Theoligonucleotide can comprise the linker. The linker can comprise achemical group. The chemical group can be reversibly or irreversiblyattached to the protein binding reagent. The chemical group can comprisea UV photocleavable group, a disulfide bond, a streptavidin, a biotin,an amine, a disulfide linkage or any combination thereof.

The location of the protein targets in the cell can vary, for example,on the cell surface or inside the cell. In some embodiments, the atleast one of the one or more protein targets is on a cell surface. Insome embodiments, the at least one of the one or more protein targets isan intracellular protein. In some embodiments, the at least one of theone or more protein targets is a transmembrane protein. In someembodiments, the at least one of the one or more protein targets is anextracellular protein. In some embodiments, the method can comprise:fixating the cell prior to contacting the cell with the first pair ofinteraction determination compositions. In some embodiments, the methodcan comprise: removing unbound interaction determination compositions ofthe first pair of interaction determination compositions. Removing theunbound interaction determination compositions can comprise washing thecell with a washing buffer. Removing the unbound interactiondetermination compositions can comprise selecting the cell using flowcytometry. In some embodiments, the method can comprise: lysing thecell.

In some embodiments, the interaction determination oligonucleotide isconfigured to be detachable or non-detachable from the protein bindingreagent. The method can comprise: detaching the interactiondetermination oligonucleotide from the protein binding reagent.Detaching the interaction determination oligonucleotide can comprisedetaching the interaction determination oligonucleotide from the proteinbinding reagent by UV photocleaving, chemical treatment, heating, enzymetreatment, or any combination thereof. The interaction determinationoligonucleotide can be not homologous to genomic sequences of the cell.The interaction determination oligonucleotide can be homologous togenomic sequences of a species. The species can be a non-mammalianspecies. The non-mammalian species can be a phage species. The phagespecies can be T7 phage, a PhiX phage, or a combination thereof. In someembodiments, the interaction determination oligonucleotide of the one ofthe first pair of interaction determination compositions comprises asequence complementary to the capture sequence. The capture sequence cancomprise a poly(dT) region. The sequence of the interactiondetermination oligonucleotide complementary to the capture sequence cancomprise a poly(dA) region. In some embodiments, the interactiondetermination oligonucleotide comprises a second barcode sequence. Theinteraction determination oligonucleotide of the other of the first pairof interaction identification compositions can comprise a binding sitefor a universal primer. The interaction determination oligonucleotidecan be associated with a detectable moiety.

In some embodiments, the protein binding reagent can be associated withtwo or more interaction determination oligonucleotides with differentinteraction determination sequences. In some embodiments, the one of theplurality of interaction determination compositions comprises a secondprotein binding reagent not associated with the interactiondetermination oligonucleotide. The protein binding reagent and thesecond protein binding reagent can be identical. The protein bindingreagent can be associated with a detectable moiety.

In some embodiments, the cell is a tumor cell or non-tumor cell. Forexample, the cell can be a mammalian cell, a bacterial cell, a viralcell, a yeast cell, a fungal cell, or any combination thereof. In someembodiments, the method comprises: contacting two or more cells with thefirst pair of interaction determination compositions, and wherein eachof the two or more cells comprises the first and the second proteintargets. At least one of the two or more cells can comprise a singlecell.

In some embodiments, the protein target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. The protein target can be, or comprise, a cell-surface protein,a cell marker, a B-cell receptor, a T-cell receptor, a majorhistocompatibility complex, a tumor antigen, a receptor, an integrin, orany combination thereof. The protein target can be, or comprise, alipid, a carbohydrate, or any combination thereof. The protein targetcan be selected from a group comprising 10-100 different proteintargets. The protein binding reagent can be associated with two or moreinteraction determination oligonucleotides with an identical sequence.

In some embodiments, the barcode comprises a cell label sequence, abinding site for a universal primer, or any combination thereof. Atleast two barcodes of the plurality of barcodes can comprise anidentical cell label sequence. In some embodiments, the plurality ofbarcodes is associated with a particle. At least one barcode theplurality of barcodes can be immobilized on the particle. At least onebarcode of the plurality of barcodes can be partially immobilized on theparticle. At least one barcode of the plurality of barcodes can beenclosed in the particle. At least one barcode of the plurality ofbarcodes can be partially enclosed in the particle. The particle can bedisruptable. The particle can comprise a bead. The particle can comprisea Sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead,a conjugated bead, a protein A conjugated bead, a protein G conjugatedbead, a protein A/G conjugated bead, a protein L conjugated bead, anoligo(dT) conjugated bead, a silica bead, a silica-like bead, ananti-biotin microbead, an anti-fluorochrome microbead, or anycombination thereof. The particle can comprise a material selected fromthe group consisting of polydimethylsiloxane (PDMS), polystyrene, glass,polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic,plastic, glass, methylstyrene, acrylic polymer, titanium, latex,Sepharose, cellulose, nylon, silicone, and any combination thereof. Theparticle can comprise a disruptable hydrogel particle. The particle canbe associated with a detectable moiety.

In some embodiments, the barcodes of the particle comprise barcodesequences selected from at least 1000 different barcode sequences. Thebarcodes of the particle can comprise barcode sequences selected fromleast 10000 different barcode sequences. The barcodes sequences of thebarcodes can comprise random sequences. The particle can comprise atleast 10000 barcodes.

In some embodiments, barcoding the interaction determinationoligonucleotides using the plurality of barcodes comprises: contactingthe plurality of barcodes with the interaction determinationoligonucleotides to generate barcodes hybridized to the interactiondetermination oligonucleotides; and extending the barcodes hybridized tothe interaction determination oligonucleotides to generate the pluralityof barcoded interaction determination oligonucleotides. Extending thebarcodes can comprise extending the barcodes using a DNA polymerase togenerate the plurality of barcoded interaction determinationoligonucleotides. Extending the barcodes can comprise extending thebarcodes using a reverse transcriptase to generate the plurality ofbarcoded interaction determination oligonucleotides. Extending thebarcodes can comprise extending the barcodes using a Moloney MurineLeukemia Virus (M-MLV) reverse transcriptase or a Taq DNA polymerase togenerate the plurality of barcoded interaction determinationoligonucleotides. Extending the barcodes can comprise displacing thebridge oligonucleotide from the ligated interaction determinationoligonucleotide.

In some embodiments, the method comprises: amplifying the plurality ofbarcoded interaction determination oligonucleotides to produce aplurality of amplicons. Amplifying the plurality of barcoded interactiondetermination oligonucleotides can comprise amplifying, using polymerasechain reaction (PCR), at least a portion of the barcode sequence and atleast a portion of the interaction determination oligonucleotide.Obtaining the sequencing data of the plurality of barcoded interactiondetermination oligonucleotides can comprise obtaining sequencing data ofthe plurality of amplicons. Obtaining the sequencing data can comprisesequencing at least a portion of the barcode sequence and at least aportion of the interaction determination oligonucleotide.

In some embodiments, obtaining sequencing data of the plurality ofbarcoded interaction determination oligonucleotides comprises obtainingpartial and/or complete sequences of the plurality of barcodedinteraction determination oligonucleotides.

In some embodiments, the plurality of barcodes comprises a plurality ofstochastic barcodes. The barcode sequence of each of the plurality ofstochastic barcodes can comprise a molecular label sequence. Themolecular label sequences of at least two stochastic barcodes of theplurality of stochastic barcodes can comprise different sequences.Barcoding the interaction determination oligonucleotides using theplurality of barcodes to create the plurality of barcoded interactiondetermination oligonucleotides can comprise stochastically barcoding theinteraction determination oligonucleotides using the plurality ofstochastic barcodes to create a plurality of stochastically barcodedinteraction determination oligonucleotides.

In some embodiments, the method comprises: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets; and obtaining sequencing data of thebarcoded targets. Barcoding the plurality of targets using the pluralityof barcodes to create the plurality of barcoded targets can comprise:contacting copies of the targets with target-binding regions of thebarcodes; and reverse transcribing the plurality targets using theplurality of barcodes to create a plurality of reverse transcribedtargets. The method can comprise: prior to obtaining the sequencing dataof the plurality of barcoded targets, amplifying the barcoded targets tocreate a plurality of amplified barcoded targets. Amplifying thebarcoded targets to generate the plurality of amplified barcoded targetscan comprise: amplifying the barcoded targets by polymerase chainreaction (PCR). Barcoding the plurality of targets of the cell using theplurality of barcodes to create the plurality of barcoded targets cancomprise stochastically barcoding the plurality of targets of the cellusing the plurality of stochastic barcodes to create a plurality ofstochastically barcoded targets.

Disclosed herein include kits for identifying interactions betweencellular components, for example protein-protein interactions. In someembodiments, the kit comprises: a first pair of interactiondetermination compositions, wherein each of the first pair ofinteraction determination compositions comprises a protein bindingreagent associated with an interaction determination oligonucleotide,wherein the protein binding reagent of one of the first pair ofinteraction determination compositions is capable of specificallybinding to a first protein target and a protein binding reagent of theother of the first pair of interaction determination compositions iscapable of specifically binding to the second protein target, whereinthe interaction determination oligonucleotide comprises an interactiondetermination sequence and a bridge oligonucleotide hybridizationregion, and wherein the interaction determination sequences of the firstpair of interaction determination compositions comprise differentsequences; and a plurality of bridge oligonucleotides each comprisingtwo hybridization regions capable of specifically binding to the bridgeoligonucleotide hybridization regions of the first pair of interactiondetermination compositions.

The length of the interaction determination sequence can vary. In someembodiments, the interaction determination sequence is at least 6nucleotides in length, 25-60 nucleotides in length, about 45 nucleotidesin length, about 50 nucleotides in length, about 100 nucleotides inlength, about 128 nucleotides in length, at least 128 nucleotides inlength, about 200-500 nucleotides in length, about 200 nucleotides inlength, at least 200 nucleotides in length, less than about 200-300nucleotides in length, about 500 nucleotides in length, or anycombination thereof.

In some embodiments, the kit further comprises: a second pair ofinteraction determination compositions, wherein each of the second pairof interaction determination compositions comprises a protein bindingreagent associated with an interaction determination oligonucleotide,wherein the protein binding reagent of one of the second pair ofinteraction determination compositions is capable of specificallybinding to a third protein target and the protein binding reagent of theother of the second pair of interaction determination compositions iscapable of specifically binding to a fourth protein target. At least oneof the third and fourth protein targets can be different from one of thefirst and second protein targets. At least one of the third and fourthprotein targets and at least one of the first and second protein targetscan be identical.

In some embodiments, the kit can comprise three or more pairs ofinteraction determination compositions. The interaction determinationsequences of at least 10 interaction determination compositions of thethree or more pairs of interaction determination compositions cancomprise different sequences. The interaction determination sequences ofat least 100 interaction determination compositions of the three or morepairs of interaction determination compositions can comprise differentsequences. The interaction determination sequences of at least 1000interaction determination compositions of the three or more pairs ofinteraction determination compositions can comprise different sequences.

In some embodiments, the bridge oligonucleotide hybridization regions oftwo interaction determination compositions of the plurality ofinteraction determination compositions comprise different sequences. Atleast one of the bridge oligonucleotide hybridization regions can becomplementary to at least one of the two hybridization regions of thebridge oligonucleotide.

In some embodiments, the protein binding reagent comprises an antibody,a tetramer, an aptamers, a protein scaffold, or a combination thereof.The interaction determination oligonucleotide can be conjugated to theprotein binding reagent through a linker. The at least one interactiondetermination oligonucleotide can comprise the linker. The linker cancomprise a chemical group. The chemical group can be reversibly orirreversibly attached to the protein binding reagent. The chemical groupcan comprise a UV photocleavable group, a disulfide bond, astreptavidin, a biotin, an amine, a disulfide linkage, or anycombination thereof. The interaction determination oligonucleotide canbe not homologous to genomic sequences of any cell of interest. The cellof interest can be a tumor cell or non-tumor cell. The cell of interestcan be a single cell, a mammalian cell, a bacterial cell, a viral cell,a yeast cell, a fungal cell, or any combination thereof.

In some embodiments, the kit further comprises: a plurality of barcodes,wherein each of the plurality of barcodes comprises a barcode sequenceand a capture sequence. The interaction determination oligonucleotide ofthe one of the first pair of interaction determination compositions cancomprise a sequence complementary to the capture sequence of at leastone barcode of a plurality of barcodes. The capture sequence cancomprise a poly(dT) region. The sequence of the interactiondetermination oligonucleotide complementary to the capture sequence ofthe barcode can comprise a poly(dA) region. The interactiondetermination oligonucleotide of the other of the first pair ofinteraction identification compositions can comprise a cell labelsequence, a binding site for a universal primer, or any combinationthereof. The plurality of barcodes can comprise a plurality ofstochastic barcodes, wherein the barcode sequence of each of theplurality of stochastic barcodes comprises a molecular label sequence,wherein the molecular label sequences of at least two stochasticbarcodes of the plurality of stochastic barcodes comprise differentsequences.

In some embodiments, the protein target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. The protein target can be, or comprise, a cell-surface protein,a cell marker, a B-cell receptor, a T-cell receptor, a majorhistocompatibility complex, a tumor antigen, a receptor, or anycombination thereof. The protein target can be selected from a groupcomprising 10-100 different protein targets. The protein binding reagentcan be associated with two or more interaction determinationoligonucleotides with an identical sequence. The protein binding reagentcan be associated with two or more interaction determinationoligonucleotides with different interaction determination sequences. Insome embodiments, the protein binding reagent can be associated with adetectable moiety.

In some embodiments, the one of the first pair of interactiondetermination compositions comprises a second protein binding reagentnot associated with the interaction determination oligonucleotide. Thefirst protein binding reagent and the second protein binding reagent canbe identical. The interaction determination oligonucleotide can beassociated with a detectable moiety.

In some embodiments, the plurality of barcodes is associated with aparticle. At least one barcode the plurality of barcodes can beimmobilized on the particle. At least one barcode of the plurality ofbarcodes can be partially immobilized on the particle. At least onebarcode of the plurality of barcodes can be enclosed in the particle. Atleast one barcode of the plurality of barcodes can be partially enclosedin the particle. The particle can be disruptable. The particle cancomprise a bead. The particle can comprise a Sepharose bead, astreptavidin bead, an agarose bead, a magnetic bead, a conjugated bead,a protein A conjugated bead, a protein G conjugated bead, a protein A/Gconjugated bead, a protein L conjugated bead, an oligo(dT) conjugatedbead, a silica bead, a silica-like bead, an anti-biotin microbead, ananti-fluorochrome microbead, or any combination thereof. The particlecan comprise a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone,and any combination thereof. The particle can comprise a disruptablehydrogel particle. The particle can be associated with a detectablemoiety.

In some embodiments, the barcodes of the particle comprise barcodesequences selected from at least 1000 different barcode sequences. Thebarcodes of the particle can comprise barcode sequences selected fromleast 10000 different barcode sequences. The barcodes sequences of thebarcodes can comprise random sequences. The particle can comprise atleast 10000 barcodes.

In some embodiments, the kit comprises: a DNA polymerase, a reversetranscriptase, a Moloney Murine Leukemia Virus (M-MLV) reversetranscriptase, a Taq DNA polymerase, or any combination thereof. The kitcan comprise a fixation agent.

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein the one or more cellscomprises one or more cellular component targets, wherein each of theplurality of sample indexing compositions comprises a cellular componentbinding reagent (e.g., an antibody) associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets (e.g., proteins), wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of at least two sample indexing compositions of theplurality of sample indexing compositions comprise different sequences;barcoding the sample indexing oligonucleotides using a plurality ofbarcodes to create a plurality of barcoded sample indexingoligonucleotides; amplifying the plurality of barcoded sample indexingoligonucleotides using a plurality of daisy-chaining amplificationprimers to create a plurality of daisy-chaining elongated amplicons;obtaining sequencing data of the plurality of daisy-chaining elongatedamplicons comprising sequencing data of the plurality of barcoded sampleindexing oligonucleotides; and identifying sample origin of at least onecell of the one or more cells based on the sample indexing sequence ofat least one barcoded sample indexing oligonucleotide of the pluralityof barcoded sample indexing oligonucleotides. The method can compriseremoving unbound sample indexing compositions of the plurality of sampleindexing compositions.

In some embodiments, the sample indexing sequence is at least 6nucleotides in length, 25-45 nucleotides in length, about 128nucleotides in length, or at least 128 nucleotides in length, or acombination thereof. The sample indexing oligonucleotide can be about 50nucleotides in length, about 100 nucleotides in length, about 200nucleotides in length, at least 200 nucleotides in length, less thanabout 200-300 nucleotides in length, about 200-500 nucleotides inlength, about 500 nucleotides in length, or a combination thereof.Sample indexing sequences of at least 10 sample indexing compositions ofthe plurality of sample indexing compositions can comprise differentsequences. Sample indexing sequences of at least 100 or 1000 sampleindexing compositions of the plurality of sample indexing compositionscan comprise different sequences.

In some embodiments, the cellular component binding reagent comprises anantibody, a tetramer, an aptamers, a protein scaffold, or a combinationthereof. The sample indexing oligonucleotide can be conjugated to thecellular component binding reagent through a linker. The oligonucleotidecan comprise the linker. The linker can comprise a chemical group. Thechemical group can be reversibly or irreversibly attached to thecellular component binding reagent. The chemical group can be selectedfrom the group consisting of a UV photocleavable group, a disulfidebond, a streptavidin, a biotin, an amine, and any combination thereof.

In some embodiments, at least one sample of the plurality of samplescomprises a single cell. The at least one of the one or more cellularcomponent targets can be on a cell surface. A sample of the plurality ofsamples can comprise a plurality of cells, a tissue, a tumor sample, orany combination thereof. The plurality of samples can comprise amammalian sample, a bacterial sample, a viral sample, a yeast sample, afungal sample, or any combination thereof.

In some embodiments, removing the unbound sample indexing compositionscomprises washing the one or more cells from each of the plurality ofsamples with a washing buffer. The method can comprise lysing the one ormore cells from each of the plurality of samples. The sample indexingoligonucleotide can be configured to be detachable or non-detachablefrom the cellular component binding reagent. The method can comprisedetaching the sample indexing oligonucleotide from the cellularcomponent binding reagent. Detaching the sample indexing oligonucleotidecan comprise detaching the sample indexing oligonucleotide from thecellular component binding reagent by UV photocleaving, chemicaltreatment (e.g., using a reducing reagent, such as dithiothreitol),heating, enzyme treatment, or any combination thereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of the cells of the plurality ofsamples. The sample indexing oligonucleotide can comprise a molecularlabel sequence, a poly(A) region, or a combination thereof. The sampleindexing oligonucleotide can comprise a sequence complementary to acapture sequence of at least one barcode of the plurality of barcodes. Atarget binding region of the barcode can comprise the capture sequence.The target binding region can comprise a poly(dT) region. The sequenceof the sample indexing oligonucleotide complementary to the capturesequence of the barcode can comprise a poly(A) tail. The sample indexingoligonucleotide can comprise a molecular label.

In some embodiments, the cellular component target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. The cellular component target can be, or comprise, acell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, or any combination thereof. The cellularcomponent target can be, or comprise, a lipid, a carbohydrate, or anycombination thereof. The cellular component target can be selected froma group comprising 10-100 different cellular component targets. Thecellular component binding reagent can be associated with two or moresample indexing oligonucleotides with an identical sequence. Thecellular component binding reagent can be associated with two or moresample indexing oligonucleotides with different sample indexingsequences. The sample indexing composition of the plurality of sampleindexing compositions can comprise a second cellular component bindingreagent not conjugated with the sample indexing oligonucleotide. Thecellular component binding reagent and the second cellular componentbinding reagent can be identical.

In some embodiments, a barcode of the plurality of barcodes comprises atarget binding region and a molecular label sequence. Molecular labelsequences of at least two barcodes of the plurality of barcodes comprisedifferent molecule label sequences. The barcode can comprise a celllabel, a binding site for a universal primer, or any combinationthereof. The target binding region can comprise a poly(dT) region.

In some embodiments, the plurality of barcodes is associated with aparticle. At least one barcode of the plurality of barcodes can beimmobilized on the particle, partially immobilized on the particle,enclosed in the particle, partially enclosed in the particle, or anycombination thereof. The particle can be degradable. The particle can bea bead. The bead can be selected from the group consisting ofstreptavidin beads, agarose beads, magnetic beads, conjugated beads,protein A conjugated beads, protein G conjugated beads, protein A/Gconjugated beads, protein L conjugated beads, oligo(dT) conjugatedbeads, silica beads, silica-like beads, anti-biotin microbead,anti-fluorochrome microbead, and any combination thereof. The particlecan comprise a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone,and any combination thereof. The particle can comprise at least 10000barcodes. In some embodiments, the barcodes of the particle can comprisemolecular label sequences selected from at least 1000 or 10000 differentmolecular label sequences. The molecular label sequences of the barcodescan comprise random sequences.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

In some embodiments, amplifying the plurality of barcoded sampleindexing oligonucleotides comprises amplifying the plurality of barcodedsample indexing oligonucleotides using polymerase chain reaction (PCR).Amplifying the plurality of barcoded sample indexing oligonucleotidescan comprise amplifying at least a portion of the molecular labelsequence and at least a portion of the sample indexing oligonucleotide.Obtaining the sequencing data of the plurality of barcoded sampleindexing oligonucleotides can comprise obtaining sequencing data of theplurality of daisy-chaining elongated amplicons. Obtaining thesequencing data can comprise sequencing at least a portion of themolecular label sequence and at least a portion of the sample indexingoligonucleotide.

In some embodiments, each daisy-chaining amplification primer of theplurality of daisy-chaining amplification primers comprises a barcodedsample indexing oligonucleotide-binding region and an overhang region,wherein the barcoded sample indexing oligonucleotide-binding region iscapable of binding to a daisy-chaining sample indexing region of thesample indexing oligonucleotide. The barcoded sample indexingoligonucleotide-binding region can be at least 20 nucleotides in length,at least 30 nucleotides in length, about 40 nucleotides in length, atleast 40 nucleotides in length, about 50 nucleotides in length, or acombination thereof. Two daisy-chaining amplification primers of theplurality of daisy-chaining amplification primers can comprise barcodedsample indexing oligonucleotide-binding regions with an identicalsequence. The plurality of daisy-chaining amplification primers cancomprise barcoded sample indexing oligonucleotide-binding regions withan identical sequence. The overhang region can be at least 50nucleotides in length, at least 100 nucleotides in length, at least 150nucleotides in length, about 150 nucleotides in length, at least 200nucleotides in length, or a combination thereof. The overhang region cancomprise a daisy-chaining amplification primer barcode sequence. Twodaisy-chaining amplification primers of the plurality of daisy-chainingamplification primers can comprise overhang regions with an identicaldaisy-chaining amplification primer barcode sequence. Two daisy-chainingamplification primers of the plurality of daisy-chaining amplificationprimers can comprise overhang regions with two daisy-chainingamplification primer barcode sequences. Overhang regions of theplurality of daisy-chaining amplification primers can comprise differentdaisy-chaining amplification primer barcode sequences. A daisy-chainingelongated amplicon of the plurality of daisy-chaining elongatedamplicons can be at least 250 nucleotides in length, at least 300nucleotides in length, at least 350 nucleotides in length, at least 400nucleotides in length, about 400 nucleotides in length, at least 450nucleotides in length, at least 500 nucleotides in length, or acombination thereof.

In some embodiments, identifying the sample origin of the at least onecell can comprise identifying sample origin of the plurality of barcodedtargets based on the sample indexing sequence of the at least onebarcoded sample indexing oligonucleotide. Barcoding the sample indexingoligonucleotides using the plurality of barcodes to create the pluralityof barcoded sample indexing oligonucleotides can comprise stochasticallybarcoding the sample indexing oligonucleotides using a plurality ofstochastic barcodes to create a plurality of stochastically barcodedsample indexing oligonucleotides.

In some embodiments, the method comprises: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label, and wherein at least two barcodes of theplurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can comprise: contacting copies of thetargets with target binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets. Prior to obtainingthe sequencing data of the plurality of barcoded targets, the method cancomprise amplifying the barcoded targets to create a plurality ofamplified barcoded targets. Amplifying the barcoded targets to generatethe plurality of amplified barcoded targets can comprise: amplifying thebarcoded targets by polymerase chain reaction (PCR). Barcoding theplurality of targets of the cell using the plurality of barcodes tocreate the plurality of barcoded targets can comprise stochasticallybarcoding the plurality of targets of the cell using a plurality ofstochastic barcodes to create a plurality of stochastically barcodedtargets.

In some embodiments, each of the plurality of sample indexingcompositions comprises the cellular component binding reagent. Thesample indexing sequences of the sample indexing oligonucleotidesassociated with two or more cellular component binding reagents can beidentical. The sample indexing sequences of the sample indexingoligonucleotides associated with two or more cellular component bindingreagents can comprise different sequences. Each of the plurality ofsample indexing compositions can comprise two or more cellular componentbinding reagents. In some embodiments, the cellular component is anantigen. The cellular component binding reagent can be an antibody.

Disclosed herein include methods for sample identification. In someembodiments, the method comprise: contacting one or more cells from eachof a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more binding targets, wherein each of theplurality of sample indexing compositions comprises a cellular componentbinding reagent associated with a sample indexing oligonucleotide,wherein the cellular component binding reagent is capable ofspecifically binding to at least one of the one or more binding targets,wherein the sample indexing oligonucleotide comprises a sample indexingsequence, and wherein sample indexing sequences of at least two sampleindexing compositions of the plurality of sample indexing compositionscomprise different sequences; barcoding the sample indexingoligonucleotides using a plurality of barcodes to create a plurality ofbarcoded sample indexing oligonucleotides; obtaining sequencing data ofthe plurality of barcoded sample indexing oligonucleotides using aplurality of daisy-chaining amplification primers; and identifyingsample origin of at least one cell of the one or more cells based on thesample indexing sequence of at least one barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides. The method can comprise removing unbound sampleindexing compositions of the plurality of sample indexing compositions.

In some embodiments, the sample indexing sequence is at least 6nucleotides in length, 25-45 nucleotides in length, about 128nucleotides in length, or at least 128 nucleotides in length, or acombination thereof. The sample indexing oligonucleotide can be about 50nucleotides in length, about 100 nucleotides in length, about 200nucleotides in length, at least 200 nucleotides in length, less thanabout 200-300 nucleotides in length, about 200-500 nucleotides inlength, about 500 nucleotides in length, or a combination thereof.Sample indexing sequences of at least 10 sample indexing compositions ofthe plurality of sample indexing compositions can comprise differentsequences. Sample indexing sequences of at least 10 sample indexingcompositions of the plurality of sample indexing compositions cancomprise different sequences. Sample indexing sequences of at least 10sample indexing compositions of the plurality of sample indexingcompositions comprise different sequences.

In some embodiments, the cellular component binding reagent comprises acell surface binding reagent, an antibody, a tetramer, an aptamers, aprotein scaffold, an integrin, or a combination thereof. The sampleindexing oligonucleotide can be conjugated to the cellular componentbinding reagent through a linker. The oligonucleotide can comprise thelinker. The linker can comprise a chemical group. The chemical group canbe reversibly or irreversibly attached to the cellular component bindingreagent. The chemical group can be selected from the group consisting ofa UV photocleavable group, a disulfide bond, a streptavidin, a biotin,an amine, and any combination thereof.

In some embodiments, at least one sample of the plurality of samplescomprises a single cell. The at least one of the one or more cellularcomponent targets can be expressed on a cell surface. A sample of theplurality of samples can comprise a plurality of cells, a tissue, atumor sample, or any combination thereof. The plurality of samples cancomprise a mammalian sample, a bacterial sample, a viral sample, a yeastsample, a fungal sample, or any combination thereof.

In some embodiments, removing the unbound sample indexing compositionscomprises washing the one or more cells from each of the plurality ofsamples with a washing buffer. The method can comprise lysing the one ormore cells from each of the plurality of samples. The sample indexingoligonucleotide can be configured to be detachable or non-detachablefrom the cellular component binding reagent. The method can comprisedetaching the sample indexing oligonucleotide from the cellularcomponent binding reagent. Detaching the sample indexing oligonucleotidecan comprise detaching the sample indexing oligonucleotide from thecellular component binding reagent by UV photocleaving, chemicaltreatment (e.g., using a reducing reagent, such as dithiothreitol),heating, enzyme treatment, or any combination thereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of the cells of the plurality ofsamples. The sample indexing oligonucleotide can comprise a molecularlabel sequence, a poly(A) region, or a combination thereof. The sampleindexing oligonucleotide can comprise a sequence complementary to acapture sequence of at least one barcode of the plurality of barcodes. Atarget binding region of the barcode can comprise the capture sequence.The target binding region can comprise a poly(dT) region. The sequenceof the sample indexing oligonucleotide complementary to the capturesequence of the barcode can comprise a poly(A) tail. The sample indexingoligonucleotide can comprise a molecular label.

In some embodiments, the cellular component target is, or comprises, acarbohydrate, a lipid, a protein, an extracellular protein, acell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, an intracellular protein, or any combinationthereof. The cellular component target can be selected from a groupcomprising 10-100 different cellular component targets. The cellularcomponent binding reagent can be associated with two or more sampleindexing oligonucleotides with an identical sequence. The cellularcomponent binding reagent can be associated with two or more sampleindexing oligonucleotides with different sample indexing sequences. Thesample indexing composition of the plurality of sample indexingcompositions can comprise a second cellular component binding reagentnot conjugated with the sample indexing oligonucleotide. The cellularcomponent binding reagent and the second cell binding reagent can beidentical.

In some embodiments, a barcode of the plurality of barcodes comprises atarget binding region and a molecular label sequence. Molecular labelsequences of at least two barcodes of the plurality of barcodes cancomprise different molecule label sequences. The barcode can comprise acell label, a binding site for a universal primer, or any combinationthereof. The target binding region can comprise a poly(dT) region.

In some embodiments, the plurality of barcodes is enclosed in aparticle. The particle can be a bead. At least one barcode of theplurality of barcodes can be immobilized on the particle, partiallyimmobilized on the particle, enclosed in the particle, partiallyenclosed in the particle, or any combination thereof. The particle canbe degradable. The bead can be selected from the group consisting ofstreptavidin beads, agarose beads, magnetic beads, conjugated beads,protein A conjugated beads, protein G conjugated beads, protein A/Gconjugated beads, protein L conjugated beads, oligo(dT) conjugatedbeads, silica beads, silica-like beads, anti-biotin microbead,anti-fluorochrome microbead, and any combination thereof. The particlecan comprise a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone,and any combination thereof. The particle can comprise at least 10000barcodes. In some embodiments, the barcodes of the particle can comprisemolecular label sequences selected from at least 1000 or 10000 differentmolecular label sequences. The molecular label sequences of the barcodescan comprise random sequences.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises amplifying the plurality ofbarcoded sample indexing oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded sample indexingoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR) at least a portion of the molecular label sequence and atleast a portion of the sample indexing oligonucleotide. Amplifying theplurality of barcoded sample indexing oligonucleotides can compriseamplifying the plurality of barcoded sample indexing oligonucleotidesusing the plurality of daisy-chaining amplification primers to producethe plurality of daisy-chaining elongated amplicons Obtaining thesequencing data of the plurality of barcoded sample indexingoligonucleotides can comprise obtaining sequencing data of the pluralityof daisy-chaining elongated amplicons. Obtaining the sequencing data cancomprise sequencing at least a portion of the molecular label sequenceand at least a portion of the sample indexing oligonucleotide.

In some embodiments, a daisy-chaining amplification primer of theplurality of daisy-chaining amplification primers comprises a barcodedsample indexing oligonucleotide-binding region and an overhang region,wherein the barcoded sample indexing oligonucleotide-binding region iscapable of binding to a daisy-chaining sample indexing region of thesample indexing oligonucleotide. The barcoded sample indexingoligonucleotide-binding region can be at least 20 nucleotides in length,at least 30 nucleotides in length, about 40 nucleotides in length, atleast 40 nucleotides in length, about 50 nucleotides in length, or acombination thereof. Two daisy-chaining amplification primers of theplurality of daisy-chaining amplification primers can comprise barcodedsample indexing oligonucleotide-binding regions with an identicalsequence. The plurality of daisy-chaining amplification primers cancomprise barcoded sample indexing oligonucleotide-binding regions withan identical sequence. The overhang region can be at least 50nucleotides in length, at least 100 nucleotides in length, at least 150nucleotides in length, about 150 nucleotides in length, at least 200nucleotides in length, or a combination thereof. The overhang region cancomprise a daisy-chaining amplification primer barcode sequence. Twodaisy-chaining amplification primers of the plurality of daisy-chainingamplification primers can comprise overhang regions with an identicaldaisy-chaining amplification primer barcode sequence. Two daisy-chainingamplification primers of the plurality of daisy-chaining amplificationprimers can comprise overhang regions with two daisy-chainingamplification primer barcode sequences. Overhang regions of theplurality of daisy-chaining amplification primers can comprise differentdaisy-chaining amplification primer barcode sequences. A daisy-chainingelongated amplicon of the plurality of daisy-chaining elongatedamplicons can be at least 250 nucleotides in length, at least 300nucleotides in length, at least 350 nucleotides in length, at least 400nucleotides in length, about 400 nucleotides in length, at least 450nucleotides in length, at least 500 nucleotides in length, or acombination thereof.

In some embodiments, identifying the sample origin of the at least onecell can comprise identifying sample origin of the plurality of barcodedtargets based on the sample indexing sequence of the at least onebarcoded sample indexing oligonucleotide. Barcoding the sample indexingoligonucleotides using the plurality of barcodes to create the pluralityof barcoded sample indexing oligonucleotides can comprise stochasticallybarcoding the sample indexing oligonucleotides using a plurality ofstochastic barcodes to create a plurality of stochastically barcodedsample indexing oligonucleotides.

In some embodiments, the method comprises: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label, and wherein at least two barcodes of theplurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can comprise: contacting copies of thetargets with target binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets. The method cancomprise: prior to obtaining the sequencing data of the plurality ofbarcoded targets, amplifying the barcoded targets to create a pluralityof amplified barcoded targets. Amplifying the barcoded targets togenerate the plurality of amplified barcoded targets can comprise:amplifying the barcoded targets by polymerase chain reaction (PCR).Barcoding the plurality of targets of the cell using the plurality ofbarcodes to create the plurality of barcoded targets can comprisestochastically barcoding the plurality of targets of the cell using aplurality of stochastic barcodes to create a plurality of stochasticallybarcoded targets.

In some embodiments, each of the plurality of sample indexingcompositions comprises the cellular component binding reagent. Thesample indexing sequences of the sample indexing oligonucleotidesassociated with two or more cellular component binding reagents can beidentical. The sample indexing sequences of the sample indexingoligonucleotides associated with two or more cellular component bindingreagents can comprise different sequences. Each of the plurality ofsample indexing compositions can comprise two or more cellular componentbinding reagents.

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more cellular component targets, whereineach of the plurality of sample indexing compositions comprises acellular component binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; andidentifying sample origin of at least one cell of the one or more cellsbased on the sample indexing sequence of at least one sample indexingoligonucleotide of the plurality of sample indexing compositions and aplurality of daisy-chaining amplification primers. Identifying thesample origin of the at least one cell can comprise: barcoding sampleindexing oligonucleotides of the plurality of sample indexingcompositions using a plurality of barcodes to create a plurality ofbarcoded sample indexing oligonucleotides; obtaining sequencing data ofthe plurality of barcoded sample indexing oligonucleotides; andidentifying the sample origin of the cell based on the sample indexingsequence of at least one barcoded sample indexing oligonucleotide of theplurality of barcoded sample indexing oligonucleotides in the sequencingdata. The method can, for example, include removing unbound sampleindexing compositions of the plurality of sample indexing compositions.

In some embodiments, the sample indexing sequence is at least 6nucleotides in length, 25-45 nucleotides in length, about 128nucleotides in length, or at least 128 nucleotides in length, or acombination thereof. The sample indexing oligonucleotide can be about 50nucleotides in length, about 100 nucleotides in length, about 200nucleotides in length, at least 200 nucleotides in length, less thanabout 200-300 nucleotides in length, about 200-500 nucleotides inlength, about 500 nucleotides in length, or a combination thereof.Sample indexing sequences of at least 10 sample indexing compositions ofthe plurality of sample indexing compositions can comprise differentsequences. Sample indexing sequences of at least 100 or 1000 sampleindexing compositions of the plurality of sample indexing compositionscan comprise different sequences.

In some embodiments, the cellular component binding reagent comprises anantibody, a tetramer, an aptamers, a protein scaffold, or a combinationthereof. The sample indexing oligonucleotide can be conjugated to thecellular component binding reagent through a linker. The oligonucleotidecan comprise the linker. The linker can comprise a chemical group. Thechemical group can be reversibly or irreversibly attached to thecellular component binding reagent. The chemical group can be selectedfrom the group consisting of a UV photocleavable group, a disulfidebond, a streptavidin, a biotin, an amine, and any combination thereof.

In some embodiments, at least one sample of the plurality of samplescomprises a single cell. The at least one of the one or more cellularcomponent targets can be expressed on a cell surface. A sample of theplurality of samples can comprise a plurality of cells, a tissue, atumor sample, or any combination thereof. The plurality of samples cancomprise a mammalian sample, a bacterial sample, a viral sample, a yeastsample, a fungal sample, or any combination thereof.

In some embodiments, removing the unbound sample indexing compositionscomprises washing the one or more cells from each of the plurality ofsamples with a washing buffer. The method can comprise lysing the one ormore cells from each of the plurality of samples. The sample indexingoligonucleotide can be configured to be detachable or non-detachablefrom the cellular component binding reagent. The method can comprisedetaching the sample indexing oligonucleotide from the cellularcomponent binding reagent. Detaching the sample indexing oligonucleotidecan comprise detaching the sample indexing oligonucleotide from thecellular component binding reagent by UV photocleaving, chemicaltreatment (e.g., using a reducing reagent, such as dithiothreitol),heating, enzyme treatment, or any combination thereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of the cells of the plurality ofsamples. The sample indexing oligonucleotide can comprise a molecularlabel sequence, a poly(A) region, or a combination thereof. The sampleindexing oligonucleotide can comprise a sequence complementary to acapture sequence of at least one barcode of the plurality of barcodes. Atarget binding region of the barcode can comprise the capture sequence.The target binding region can comprise a poly(dT) region. The sequenceof the sample indexing oligonucleotide complementary to the capturesequence of the barcode can comprise a poly(A) tail. The sample indexingoligonucleotide can comprise a molecular label.

In some embodiments, the cellular component target is, or comprises, acarbohydrate, a lipid, a protein, an extracellular protein, acell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an intracellular protein, or any combination thereof. Thecellular component target can be selected from a group comprising 10-100different cellular component targets. The cellular component bindingreagent can be associated with two or more sample indexingoligonucleotides with an identical sequence. The cellular componentbinding reagent can associated with two or more sample indexingoligonucleotides with different sample indexing sequences. The sampleindexing composition of the plurality of sample indexing compositionscan comprise a second cellular component binding reagent not conjugatedwith the sample indexing oligonucleotide. The cellular component bindingreagent and the second cellular component binding reagent can beidentical.

In some embodiments, identifying the sample origin of the at least onecell comprises: barcoding sample indexing oligonucleotides of theplurality of sample indexing compositions using a plurality of barcodesto create a plurality of barcoded sample indexing oligonucleotides;obtaining sequencing data of the plurality of barcoded sample indexingoligonucleotides; and identifying the sample origin of the cell based onthe sample indexing sequence of at least one barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides.

In some embodiments, a barcode of the plurality of barcodes comprises atarget binding region and a molecular label sequence. Molecular labelsequences of at least two barcodes of the plurality of barcodes cancomprise different molecule label sequences. The barcode can comprise acell label, a binding site for a universal primer, or any combinationthereof. The target binding region can comprise a poly(dT) region.

In some embodiments, the plurality of barcodes is immobilized on aparticle. At least one barcode of the plurality of barcodes can beimmobilized on the particle, partially immobilized on the particle,enclosed in the particle, partially enclosed in the particle, or anycombination thereof. The particle can be degradable. The particle can bea bead. The bead can be selected from the group consisting ofstreptavidin beads, agarose beads, magnetic beads, conjugated beads,protein A conjugated beads, protein G conjugated beads, protein A/Gconjugated beads, protein L conjugated beads, oligo(dT) conjugatedbeads, silica beads, silica-like beads, anti-biotin microbead,anti-fluorochrome microbead, and any combination thereof. The particlecan comprise a material selected from the group consisting ofpolydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose,gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene,acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone,and any combination thereof. The particle can comprise at least 10000barcodes. In some embodiments, the barcodes of the particle can comprisemolecular label sequences selected from at least 1000 or 10000 differentmolecular label sequences. The molecular label sequences of the barcodescan comprise random sequences.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes can comprise: contacting the pluralityof barcodes with the sample indexing oligonucleotides to generatebarcodes hybridized to the sample indexing oligonucleotides; andextending the barcodes hybridized to the sample indexingoligonucleotides to generate the plurality of barcoded sample indexingoligonucleotides. Extending the barcodes can comprise extending thebarcodes using a DNA polymerase to generate the plurality of barcodedsample indexing oligonucleotides. Extending the barcodes can compriseextending the barcodes using a reverse transcriptase to generate theplurality of barcoded sample indexing oligonucleotides.

In some embodiments, amplifying the plurality of barcoded sampleindexing oligonucleotides comprises amplifying the plurality of barcodedsample indexing oligonucleotides using polymerase chain reaction (PCR).Amplifying the plurality of barcoded sample indexing oligonucleotidescan comprise amplifying at least a portion of the molecular labelsequence and at least a portion of the sample indexing oligonucleotide.Amplifying the plurality of barcoded sample indexing oligonucleotidescan comprise amplifying the plurality of barcoded sample indexingoligonucleotides using the plurality of daisy-chaining amplificationprimers to produce a plurality of daisy-chaining elongated amplicons.Obtaining the sequencing data of the plurality of barcoded sampleindexing oligonucleotides can comprise obtaining sequencing data of theplurality of daisy-chaining elongated amplicons. Obtaining thesequencing data can comprise sequencing at least a portion of themolecular label sequence and at least a portion of the sample indexingoligonucleotide.

In some embodiments, a daisy-chaining amplification primer of theplurality of daisy-chaining amplification primers comprises a barcodedsample indexing oligonucleotide-binding region and an overhang region,wherein the barcoded sample indexing oligonucleotide-binding region iscapable of binding to a daisy-chaining sample indexing region of thesample indexing oligonucleotide. The barcoded sample indexingoligonucleotide-binding region can be at least 20 nucleotides in length,at least 30 nucleotides in length, about 40 nucleotides in length, atleast 40 nucleotides in length, about 50 nucleotides in length, or acombination thereof. Two daisy-chaining amplification primers of theplurality of daisy-chaining amplification primers can comprise barcodedsample indexing oligonucleotide-binding regions with an identicalsequence. The plurality of daisy-chaining amplification primers cancomprise barcoded sample indexing oligonucleotide-binding regions withan identical sequence. The overhang region can be at least 50nucleotides in length, at least 100 nucleotides in length, at least 150nucleotides in length, about 150 nucleotides in length, at least 200nucleotides in length, or a combination thereof. The overhang region cancomprise a daisy-chaining amplification primer barcode sequence. Twodaisy-chaining amplification primers of the plurality of daisy-chainingamplification primers can comprise overhang regions with an identicaldaisy-chaining amplification primer barcode sequence. Two daisy-chainingamplification primers of the plurality of daisy-chaining amplificationprimers can comprise overhang regions with two daisy-chainingamplification primer barcode sequences. Overhang regions of theplurality of daisy-chaining amplification primers can comprise differentdaisy-chaining amplification primer barcode sequences. A daisy-chainingelongated amplicon of the plurality of daisy-chaining elongatedamplicons can be at least 250 nucleotides in length, at least 300nucleotides in length, at least 350 nucleotides in length, at least 400nucleotides in length, at least 450 nucleotides in length, at least 500nucleotides in length, or a combination thereof. A daisy-chainingelongated amplicon of the plurality of daisy-chaining elongatedamplicons can be about 200 nucleotides in length, about 250 nucleotidesin length, about 300 nucleotides in length, about 350 nucleotides inlength, about 400 nucleotides in length, about 450 nucleotides inlength, about 500 nucleotides in length, about 600 nucleotides inlength, or a range between any two of these values.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes to create the plurality of barcodedsample indexing oligonucleotides comprises stochastically barcoding thesample indexing oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded sampleindexing oligonucleotides. Identifying the sample origin of the at leastone cell can comprise identifying the presence or absence of the sampleindexing sequence of at least one sample indexing oligonucleotide of theplurality of sample indexing compositions. Identifying the presence orabsence of the sample indexing sequence can comprise: replicating the atleast one sample indexing oligonucleotide to generate a plurality ofreplicated sample indexing oligonucleotides; obtaining sequencing dataof the plurality of replicated sample indexing oligonucleotides; andidentifying the sample origin of the cell based on the sample indexingsequence of a replicated sample indexing oligonucleotide of theplurality of sample indexing oligonucleotides that correspond to theleast one barcoded sample indexing oligonucleotide in the sequencingdata.

In some embodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, ligating a replicating adaptorto the at least one barcoded sample indexing oligonucleotide.Replicating the at least one barcoded sample indexing oligonucleotidecan comprise replicating the at least one barcoded sample indexingoligonucleotide using the replicating adaptor ligated to the at leastone barcoded sample indexing oligonucleotide to generate the pluralityof replicated sample indexing oligonucleotides.

In some embodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, contacting a capture probewith the at least one sample indexing oligonucleotide to generate acapture probe hybridized to the sample indexing oligonucleotide; andextending the capture probe hybridized to the sample indexingoligonucleotide to generate a sample indexing oligonucleotide associatedwith the capture probe. Replicating the at least one sample indexingoligonucleotide can comprise replicating the sample indexingoligonucleotide associated with the capture probe to generate theplurality of replicated sample indexing oligonucleotides.

In some embodiments, the method comprises: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label, and wherein at least two barcodes of theplurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Identifying thesample origin of the at least one barcoded sample indexingoligonucleotide can comprise identifying the sample origin of theplurality of barcoded targets based on the sample indexing sequence ofthe at least one barcoded sample indexing oligonucleotide. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can comprise: contacting copies of thetargets with target binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets. The method cancomprise: prior to obtaining the sequencing data of the plurality ofbarcoded targets, amplifying the barcoded targets to create a pluralityof amplified barcoded targets. Amplifying the barcoded targets togenerate the plurality of amplified barcoded targets can comprise:amplifying the barcoded targets by polymerase chain reaction (PCR).Barcoding the plurality of targets of the cell using the plurality ofbarcodes to create the plurality of barcoded targets can comprisestochastically barcoding the plurality of targets of the cell using aplurality of stochastic barcodes to create a plurality of stochasticallybarcoded targets.

In some embodiments, each of the plurality of sample indexingcompositions comprises the cellular component binding reagent. Thesample indexing sequences of the sample indexing oligonucleotidesassociated with the two or more cellular component binding reagents canbe identical. The sample indexing sequences of the sample indexingoligonucleotides associated with the two or more cellular componentbinding reagents can comprise different sequences. Each of the pluralityof sample indexing compositions can comprise the two or more cellularcomponent binding reagents.

Disclosed herein includes a kit comprising: a plurality of sampleindexing compositions; and a plurality of daisy-chaining amplificationprimers. Each of the plurality of sample indexing compositions cancomprise two or more cellular component binding reagents. Each of thetwo or more cellular component binding reagents can be associated with asample indexing oligonucleotide. At least one of the two or morecellular component binding reagents can be capable of specificallybinding to at least one cellular component target. The sample indexingoligonucleotide can comprise a sample indexing sequence for identifyingsample origin of one or more cells of a sample. The sample indexingoligonucleotide can comprise a daisy-chaining amplification primerbinding sequence. Sample indexing sequences of at least two sampleindexing compositions of the plurality of sample indexing compositionscan comprise different sequences.

A daisy-chaining amplification primer of the plurality of daisy-chainingamplification primers can comprise a barcoded sample indexingoligonucleotide-binding region and an overhang region, wherein thebarcoded sample indexing oligonucleotide-binding region is capable ofbinding to a daisy-chaining sample indexing region of the sampleindexing oligonucleotide. The barcoded sample indexingoligonucleotide-binding region can be at least 20 nucleotides in length,at least 30 nucleotides in length, about 40 nucleotides in length, atleast 40 nucleotides in length, about 50 nucleotides in length, or acombination thereof. Two daisy-chaining amplification primers of theplurality of daisy-chaining amplification primers can comprise barcodedsample indexing oligonucleotide-binding regions with an identicalsequence. Two daisy-chaining amplification primers of the plurality ofdaisy-chaining amplification primers can comprise barcoded sampleindexing oligonucleotide-binding regions with different sequences. Thebarcoded sample indexing oligonucleotide-binding regions can comprisethe daisy-chaining amplification primer binding sequence, a complementthereof, a reverse complement thereof, or a combination thereof.Daisy-chaining amplification primer binding sequences of at least twosample indexing compositions of the plurality of sample indexingcompositions can comprise an identical sequence. Daisy-chainingamplification primer binding sequences of at least two sample indexingcompositions of the plurality of sample indexing compositions comprisedifferent sequences. The overhang region can be at least 50 nucleotidesin length, at least 100 nucleotides in length, at least 150 nucleotidesin length, about 150 nucleotides in length, or a combination thereof.The overhang region can be at least 200 nucleotides in length. Theoverhang region can comprise a daisy-chaining amplification primerbarcode sequence. Two daisy-chaining amplification primers of theplurality of daisy-chaining amplification primers can comprise overhangregions with an identical daisy-chaining amplification primer barcodesequence. Two daisy-chaining amplification primers of the plurality ofdaisy-chaining amplification primers can comprise overhang regions withtwo daisy-chaining amplification primer barcode sequences. Overhangregions of the plurality of daisy-chaining amplification primers cancomprise different daisy-chaining amplification primer barcodesequences.

In some embodiments, the sample indexing sequence is at least 6nucleotides in length, 25-45 nucleotides in length, about 128nucleotides in length, or at least 128 nucleotides in length, or acombination thereof. The sample indexing oligonucleotide can be about 50nucleotides in length, about 100 nucleotides in length, about 200nucleotides in length, at least 200 nucleotides in length, less thanabout 200-300 nucleotides in length, about 200-500 nucleotides inlength, about 500 nucleotides in length, or a combination thereof.Sample indexing sequences of at least 10, 100, or 1000 sample indexingcompositions of the plurality of sample indexing compositions comprisedifferent sequences.

In some embodiments, the cellular component binding reagent comprises anantibody, a tetramer, an aptamers, a protein scaffold, or a combinationthereof. The sample indexing oligonucleotide can be conjugated to thecellular component binding reagent through a linker. The at least onesample indexing oligonucleotide can comprise the linker. The linker cancomprise a chemical group. The chemical group can be reversibly orirreversibly attached to the molecule of the cellular component bindingreagent. The chemical group can be selected from the group consisting ofa UV photocleavable group, a disulfide bond, a streptavidin, a biotin,an amine, and any combination thereof.

In some embodiments, the sample indexing oligonucleotide is nothomologous to genomic sequences of a species. The sample indexingoligonucleotide can comprise a molecular label sequence, a poly(A)region, or a combination thereof. In some embodiments, at least onesample of the plurality of samples can comprise a single cell, aplurality of cells, a tissue, a tumor sample, or any combinationthereof. The sample can comprise a mammalian sample, a bacterial sample,a viral sample, a yeast sample, a fungal sample, or any combinationthereof.

In some embodiments, the cellular component target is, or comprises, acell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, or any combination thereof. The cellular component target canbe selected from a group comprising 10-100 different cellular componenttargets. The cellular component binding reagent can be associated withtwo or more sample indexing oligonucleotides with an identical sequence.The cellular component binding reagent can be associated with two ormore sample indexing oligonucleotides with different sample indexingsequences. The sample indexing composition of the plurality of sampleindexing compositions can comprise a second cellular component bindingreagent not conjugated with the sample indexing oligonucleotide. Thecellular component binding reagent and the second cellular componentbinding reagent can be identical.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a non-limiting exemplary stochastic barcode.

FIG. 2 shows a non-limiting exemplary workflow of stochastic barcodingand digital counting.

FIG. 3 is a schematic illustration showing a non-limiting exemplaryprocess for generating an indexed library of the stochastically barcodedtargets from a plurality of targets.

FIG. 4 shows a schematic illustration of an exemplary protein bindingreagent (antibody illustrated here) conjugated with an oligonucleotidecomprising a unique identifier for the protein binding reagent.

FIG. 5 shows a schematic illustration of an exemplary binding reagent(antibody illustrated here) conjugated with an oligonucleotidecomprising a unique identifier for sample indexing to determine cellsfrom the same or different samples.

FIG. 6 shows a schematic illustration of an exemplary workflow usingoligonucleotide-conjugated antibodies to determine protein expressionand gene expression simultaneously in a high throughput manner.

FIG. 7 shows a schematic illustration of an exemplary workflow of usingoligonucleotide-conjugated antibodies for sample indexing.

FIGS. 8A1 and 8A2 show a schematic illustration of an exemplary workflowof daisy-chaining a barcoded oligonucleotide. FIGS. 8B1 and 8B2 show aschematic illustration of another exemplary workflow of daisy-chaining abarcoded oligonucleotide.

FIGS. 9A-9E show non-limiting exemplary schematic illustrations ofparticles functionalized with oligonucleotides.

FIG. 10A is a schematic illustration of an exemplary workflow of usingparticles functionalized with oligonucleotides for single cellsequencing control. FIG. 10B is a schematic illustration of anotherexemplary workflow of using particles functionalized witholigonucleotides for single cell sequencing control.

FIG. 11 shows a schematic illustration of an exemplary workflow of usingcontrol oligonucleotide-conjugated antibodies for determining singlecell sequencing efficiency.

FIG. 12 shows another schematic illustration of an exemplary workflow ofusing control oligonucleotide-conjugated antibodies for determiningsingle cell sequencing efficiency.

FIGS. 13A-13C are plots showing that control oligonucleotides can beused for cell counting.

FIGS. 14A-14F show a schematic illustration of an exemplary workflow ofdetermining interactions between cellular components (e.g., proteins)using a pair of interaction determination compositions.

FIGS. 15A-15D show non-limiting exemplary designs of oligonucleotidesfor determining protein expression and gene expression simultaneouslyand for sample indexing.

FIG. 16 shows a schematic illustration of a non-limiting exemplaryoligonucleotide sequence for determining protein expression and geneexpression simultaneously and for sample indexing.

FIGS. 17A-17F are non-limiting exemplary tSNE projection plots showingresults of using oligonucleotide-conjugated antibodies to measure CD4protein expression and gene expression simultaneously in a highthroughput manner.

FIGS. 18A-18F are non-limiting exemplary bar charts showing theexpressions of CD4 mRNA and protein in CD4 T cells, CD8 T cells, andMyeloid cells.

FIG. 19 is a non-limiting exemplary bar chart showing that, with similarsequencing depth, detection sensitivity for CD4 protein level increasedwith higher ratios of antibody:oligonucleotide, with the 1:3 ratioperforming better than the 1:1 and 1:2 ratios.

FIGS. 20A-20D are plots showing the CD4 protein expression on cellsurface of cells sorted using flow cytometry.

FIG. 21A-21F are non-limiting exemplary bar charts showing theexpressions of CD4 mRNA and protein in CD4 T cells, CD8 T cells, andMyeloid cells of two samples.

FIG. 22 is a non-limiting exemplary bar chart showing detectionsensitivity for CD4 protein level determined using different samplepreparation protocols with an antibody:oligonucleotide ratio of 1:3.

FIG. 23 shows a non-limiting exemplary experimental design forperforming sample indexing and determining the effects of the lengths ofthe sample indexing oligonucleotides and the cleavability of sampleindexing oligonucleotides on sample indexing.

FIGS. 24A-24C are non-limiting exemplary tSNE plots showing that thethree types of anti-CD147 antibody conjugated with different sampleindexing oligonucleotides (cleavable 95mer, non-cleavable 95mer, andcleavable 200mer) can be used for determining the protein expressionlevel of CD147.

FIG. 25 is a non-limiting exemplary tSNE plot with an overlay of GAPDHexpression per cell.

FIGS. 26A-26C are non-limiting exemplary histograms showing the numbersof molecules of sample indexing oligonucleotides detected using thethree types of sample indexing oligonucleotides.

FIGS. 27A-27C are non-limiting exemplary plots and bar charts showingthat CD147 expression was higher in dividing cells.

FIGS. 28A-28C are non-limiting exemplary tSNE projection plots showingthat sample indexing can be used to identify cells of different samples.

FIGS. 29A-29C are non-limiting exemplary histograms of the sampleindexing sequences per cell based on the numbers of molecules of thesample indexing oligonucleotides determined.

FIGS. 30A-30D are non-limiting exemplary plots comparing annotations ofcell types determined based on mRNA expression of CD3D (for Jurkatcells) and JCHAIN (for Ramos cells) and sample indexing of Jurkat andRamos cells.

FIGS. 31A-31C are non-limiting tSNE projection plots of the mRNAexpression profiles of Jurkat and Ramos cells with overlays of the mRNAexpressions of CD3D and JCHAIN (FIG. 31A), JCHAIN (FIG. 31B), and CD3D(FIG. 31C).

FIG. 32 is a non-limiting exemplary tSNE projection plot of expressionprofiles of Jurkat and Ramos cells with an overlay of the cell typesdetermined using sample indexing with a DBEC cutoff of 250.

FIGS. 33A-33C are non-limiting exemplary bar charts of the numbers ofmolecules of sample indexing oligonucleotides per cell for Ramos &Jurkat cells (FIG. 33), Ramos cells (FIG. 33B), and Jurkat cells (FIG.33C) that were not labeled or labeled with “Short 3” sample indexingoligonucleotides, “Short 2” & “Short 3” sample indexingoligonucleotides, and “Short 2” sample indexing oligonucleotides.

FIGS. 34A-34C are non-limiting exemplary plots showing that less than 1%of single cells were labeled with both the “Short 2” and “Short 3”sample indexing oligonucleotides.

FIGS. 35A-35C are non-limiting exemplary tSNE plots showing batcheffects on expression profiles of Jurkat and Ramos cells among samplesprepared using different flowcells as outlined in FIG. 23.

FIGS. 36A1-36A9, 36B and 36C are non-limiting exemplary plots showingdetermination of an optimal dilution of an antibody stock using dilutiontitration.

FIG. 37 shows a non-limiting exemplary experimental design fordetermining a staining concentration of oligonucleotide-conjugatedantibodies such that the antibody oligonucleotides account for a desiredpercentage of total reads in sequencing data.

FIGS. 38A-38D are non-limiting exemplary bioanalyzer traces showingpeaks (indicated by arrows) consistent with the expected size of theantibody oligonucleotide.

FIGS. 39A1-39A3 and 39B1-39B3 are non-limiting exemplary histogramsshowing the numbers of molecules of antibody oligonucleotides detectedfor samples stained with different antibody dilutions and differentpercentage of the antibody molecules conjugated with the antibodyoligonucleotides (“hot antibody”).

FIGS. 40A-40C are non-limiting exemplary plots showing thatoligonucleotide-conjugated anti-CD147 antibody molecules can be used tolabel various cell types. The cell types were determined using theexpression profiles of 488 genes in a blood panel (FIG. 40A). The cellswere stained with a mixture of 10% hot antibody:90% cold antibodyprepared using a 1:100 diluted stock, resulting in a clear signal in ahistogram showing the numbers of molecules of antibody oligonucleotidesdetected (FIG. 40B). The labeling of the various cell types by theantibody oligonucleotide is shown in FIG. 40C.

FIGS. 41A-41C are non-limiting exemplary plots showing thatoligonucleotide-conjugated anti-CD147 antibodies can be used to labelvarious cell types. The cell types were determined using the expressionprofiles of 488 genes in a blood panel (FIG. 41A). The cells werestained with a mixture of 1% hot antibody:99% cold antibody preparedusing a 1:100 diluted stock, resulting in no clear signal in a histogramshowing the numbers of molecules of antibody oligonucleotides detected(FIG. 41B). The labeling of the various cell types by the antibodyoligonucleotide is shown in FIG. 41C.

FIGS. 42A-42C are non-limiting exemplary plots showing thatoligonucleotide-conjugated anti-CD147 antibody molecules can be used tolabel various cell types. The cell types were determined using theexpression profiles of 488 genes in a blood panel (FIG. 42A). The cellswere stained with a 1:800 diluted stock, resulting in a clear signal ina histogram showing the numbers of molecules of antibodyoligonucleotides detected (FIG. 42B). The labeling of the various celltypes by the antibody oligonucleotide is shown in FIG. 42C.

FIGS. 43A-43B are plots showing the composition of control particleoligonucleotides in a staining buffer and control particleoligonucleotides associated with control particles detected using theworkflow illustrated in FIG. 10.

FIGS. 44A-44B are brightfield images of cells (FIG. 44A, white circles)and control particles (FIG. 44B, black circles) in a hemocytometer.

FIGS. 45A-45B are phase contrast (FIG. 45A, 10X) and fluorescent (FIG.45B, 10X) images of control particles bound tooligonucleotide-conjugated antibodies associated with fluorophores.

FIG. 46 is an image showing cells and a control particle being loadedinto microwells of a cartridge.

FIGS. 47A-47C are plots showing using an antibody cocktail for sampleindexing can increase labeling sensitivity.

FIG. 48 is a non-limiting exemplary plot showing that multiplets can beidentified and removed from sequencing data using sample indexing.

FIG. 49A is a non-limiting exemplary tSNE projection plot of expressionprofiles of CD45+ single cells from 12 samples of six tissues from twomice identified as singlets or multiplets using sample indexingoligonucleotides.

FIG. 49B is a non-limiting exemplary tSNE projection plot of expressionprofiles of CD45+ single cells of 12 samples of six tissues from twomice with multiplets identified using sample indexing oligonucleotidesand shown in FIG. 49A removed.

FIG. 50A is a non-limiting exemplary tSNE projection plot of expressionprofiles of CD45+ single cells from two mice with multiplets identifiedusing sample indexing oligonucleotides removed.

FIGS. 50B and 50C are non-limiting exemplary pie charts showing thatafter multiplet expression profiles were removed, the two mice, whichwere biological replicates, exhibited similar expression profiles.

FIGS. 51A-51F are non-limiting exemplary pie charts showing immune cellprofiles of six different tissues with multiplet expression profiles insequencing data identified and removed using sample indexingoligonucleotides.

FIGS. 52A-52C are non-limiting exemplary graphs showing expressionprofiles of macrophages, T cells, and B cells from six different tissuesafter multiplet expression profiles in sequencing data identified andremoved using sample indexing oligonucleotides.

FIGS. 53A-53D are non-limiting exemplary plots comparing macrophages inthe colon and spleen.

FIG. 54 is a non-limiting exemplary plot showing that tagging cells withsample indexing compositions did not alter mRNA expression profiles inPBMCs.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe Figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein and made part of the disclosure herein.

All patents, published patent applications, other publications, andsequences from GenBank, and other databases referred to herein areincorporated by reference in their entirety with respect to the relatedtechnology.

Quantifying small numbers of nucleic acids, for example messengerribonucleotide acid (mRNA) molecules, is clinically important fordetermining, for example, the genes that are expressed in a cell atdifferent stages of development or under different environmentalconditions. However, it can also be very challenging to determine theabsolute number of nucleic acid molecules (e.g., mRNA molecules),especially when the number of molecules is very small. One method todetermine the absolute number of molecules in a sample is digitalpolymerase chain reaction (PCR). Ideally, PCR produces an identical copyof a molecule at each cycle. However, PCR can have disadvantages suchthat each molecule replicates with a stochastic probability, and thisprobability varies by PCR cycle and gene sequence, resulting inamplification bias and inaccurate gene expression measurements.Stochastic barcodes with unique molecular labels (also referred to asmolecular indexes (MIs)) can be used to count the number of moleculesand correct for amplification bias. Stochastic barcoding such as thePrecise™ assay (Cellular Research, Inc. (Palo Alto, Calif.)) can correctfor bias induced by PCR and library preparation steps by using molecularlabels (MLs) to label mRNAs during reverse transcription (RT).

The Precise™ assay can utilize a non-depleting pool of stochasticbarcodes with large number, for example 6561 to 65536, unique molecularlabels on poly(T) oligonucleotides to hybridize to all poly(A)-mRNAs ina sample during the RT step. A stochastic barcode can comprise auniversal PCR priming site. During RT, target gene molecules reactrandomly with stochastic barcodes. Each target molecule can hybridize toa stochastic barcode resulting to generate stochastically barcodedcomplementary ribonucleotide acid (cDNA) molecules). After labeling,stochastically barcoded cDNA molecules from microwells of a microwellplate can be pooled into a single tube for PCR amplification andsequencing. Raw sequencing data can be analyzed to produce the number ofreads, the number of stochastic barcodes with unique molecular labels,and the numbers of mRNA molecules.

Methods for determining mRNA expression profiles of single cells can beperformed in a massively parallel manner. For example, the Precise™assay can be used to determine the mRNA expression profiles of more than10000 cells simultaneously. The number of single cells (e.g., 100s or1000s of singles) for analysis per sample can be lower than the capacityof the current single cell technology. Pooling of cells from differentsamples enables improved utilization of the capacity of the currentsingle technology, thus lowering reagents wasted and the cost of singlecell analysis. The disclosure provides methods of sample indexing fordistinguishing cells of different samples for cDNA library preparationfor cell analysis, such as single cell analysis. Pooling of cells fromdifferent samples can minimize the variations in cDNA librarypreparation of cells of different samples, thus enabling more accuratecomparisons of different samples.

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more antigen targets, wherein each of theplurality of sample indexing compositions comprises a protein bindingreagent associated with a sample indexing oligonucleotide, wherein theprotein binding reagent is capable of specifically binding to at leastone of the one or more antigen targets, wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of at least two sample indexing compositions of theplurality of sample indexing compositions comprise different sequences;removing unbound sample indexing compositions of the plurality of sampleindexing compositions; barcoding (e.g., stochastically barcoding) thesample indexing oligonucleotides using a plurality of barcodes (e.g.,stochastic barcodes) to create a plurality of barcoded sample indexingoligonucleotides (e.g., stochastically barcoded sample indexingoligonucleotides); obtaining sequencing data of the plurality ofbarcoded sample indexing oligonucleotides; and identifying sample originof at least one cell of the one or more cells based on the sampleindexing sequence of at least one barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides.

In some embodiments, the method for sample identification disclosedherein comprises: contacting one or more cells from each of a pluralityof samples with a sample indexing composition of a plurality of sampleindexing compositions, wherein each of the one or more cells comprisesone or more cellular component targets, wherein each of the plurality ofsample indexing compositions comprises a cellular component bindingreagent associated with a sample indexing oligonucleotide, wherein thecellular component binding reagent is capable of specifically binding toat least one of the one or more cellular component targets, wherein thesample indexing oligonucleotide comprises a sample indexing sequence,and wherein sample indexing sequences of at least two sample indexingcompositions of the plurality of sample indexing compositions comprisedifferent sequences; removing unbound sample indexing compositions ofthe plurality of sample indexing compositions; barcoding (e.g.,stochastically barcoding) the sample indexing oligonucleotides using aplurality of barcodes (e.g., stochastic barcodes) to create a pluralityof barcoded sample indexing oligonucleotides (e.g., stochasticallybarcoded sample indexing oligonucleotides); obtaining sequencing data ofthe plurality of barcoded sample indexing oligonucleotides; andidentifying sample origin of at least one cell of the one or more cellsbased on the sample indexing sequence of at least one barcoded sampleindexing oligonucleotide of the plurality of barcoded sample indexingoligonucleotides.

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more antigen targets, wherein each of theplurality of sample indexing compositions comprises a protein bindingreagent associated with a sample indexing oligonucleotide, wherein theprotein binding reagent is capable of specifically binding to at leastone of the one or more antigen targets, wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of at least two sample indexing compositions of theplurality of sample indexing compositions comprise different sequences;removing unbound sample indexing compositions of the plurality of sampleindexing compositions; and identifying sample origin of at least onecell of the one or more cells based on the sample indexing sequence ofat least one sample indexing oligonucleotide of the plurality of sampleindexing compositions.

Disclosed herein is a plurality of sample indexing compositions. Each ofthe plurality of sample indexing compositions can comprise a proteinbinding reagent associated with a sample indexing oligonucleotide. Theprotein binding reagent can be capable of specifically binding to atleast one antigen target. The sample indexing oligonucleotide cancomprise a sample indexing sequence for identifying sample origin of oneor more cells of a sample. Sample indexing sequences of at least twosample indexing compositions of the plurality of sample indexingcompositions can comprise different sequences.

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein the one or more cellscomprises one or more cellular component targets, wherein each of theplurality of sample indexing compositions comprises a cellular componentbinding reagent (e.g., an antibody) associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets (e.g., proteins), wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of at least two sample indexing compositions of theplurality of sample indexing compositions comprise different sequences;barcoding the sample indexing oligonucleotides using a plurality ofbarcodes to create a plurality of barcoded sample indexingoligonucleotides; amplifying the plurality of barcoded sample indexingoligonucleotides using a plurality of daisy-chaining amplificationprimers to create a plurality of daisy-chaining elongated amplicons;obtaining sequencing data of the plurality of daisy-chaining elongatedamplicons comprising sequencing data of the plurality of barcoded sampleindexing oligonucleotides; and identifying sample origin of at least onecell of the one or more cells based on the sample indexing sequence ofat least one barcoded sample indexing oligonucleotide of the pluralityof barcoded sample indexing oligonucleotides. The method can compriseremoving unbound sample indexing compositions of the plurality of sampleindexing compositions.

In some embodiments, the sample indexing methods, kits, and compositionsdisclosed herein can increase sample throughput (e.g., for rare samplesof low cell number, hard to isolate cells, and heterogeneous cells),lower reagent costs, reduce technical errors and batch effects byperforming library preparation in a single tube reaction, and/oridentify inter-sample multiplet cells during data analysis. In someembodiments, cells of tissues from different lymphoid organs andnon-lymphoid organs can be tagged using different sample indexingcompositions to, for example, increase sample throughput and reduce, orminimize, batch effects. In some embodiments, immune defense and tissuehomeostasis and functions can be investigated using the methods, kits,and compositions of the disclosure.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. See, e.g., Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley& Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.1989). For purposes of the present disclosure, the following terms aredefined below.

As used herein, the term “adaptor” can mean a sequence to facilitateamplification or sequencing of associated nucleic acids. The associatednucleic acids can comprise target nucleic acids. The associated nucleicacids can comprise one or more of spatial labels, target labels, samplelabels, indexing label, or barcode sequences (e.g., molecular labels).The adapters can be linear. The adaptors can be pre-adenylated adapters.The adaptors can be double- or single-stranded. One or more adaptor canbe located on the 5′ or 3′ end of a nucleic acid. When the adaptorscomprise known sequences on the 5′ and 3′ ends, the known sequences canbe the same or different sequences. An adaptor located on the 5′ and/or3′ ends of a polynucleotide can be capable of hybridizing to one or moreoligonucleotides immobilized on a surface. An adapter can, in someembodiments, comprise a universal sequence. A universal sequence can bea region of nucleotide sequence that is common to two or more nucleicacid molecules. The two or more nucleic acid molecules can also haveregions of different sequence. Thus, for example, the 5′ adapters cancomprise identical and/or universal nucleic acid sequences and the 3′adapters can comprise identical and/or universal sequences. A universalsequence that may be present in different members of a plurality ofnucleic acid molecules can allow the replication or amplification ofmultiple different sequences using a single universal primer that iscomplementary to the universal sequence. Similarly, at least one, two(e.g., a pair) or more universal sequences that may be present indifferent members of a collection of nucleic acid molecules can allowthe replication or amplification of multiple different sequences usingat least one, two (e.g., a pair) or more single universal primers thatare complementary to the universal sequences. Thus, a universal primerincludes a sequence that can hybridize to such a universal sequence. Thetarget nucleic acid sequence-bearing molecules may be modified to attachuniversal adapters (e.g., non-target nucleic acid sequences) to one orboth ends of the different target nucleic acid sequences. The one ormore universal primers attached to the target nucleic acid can providesites for hybridization of universal primers. The one or more universalprimers attached to the target nucleic acid can be the same or differentfrom each other.

As used herein, an antibody can be a full-length (e.g., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

In some embodiments, an antibody is a functional antibody fragment. Forexample, an antibody fragment can be a portion of an antibody such asF(ab′)2, Fab′, Fab, Fv, sFv and the like. An antibody fragment can bindwith the same antigen that is recognized by the full-length antibody. Anantibody fragment can include isolated fragments consisting of thevariable regions of antibodies, such as the “Fv” fragments consisting ofthe variable regions of the heavy and light chains and recombinantsingle chain polypeptide molecules in which light and heavy variableregions are connected by a peptide linker (“scFv proteins”). Exemplaryantibodies can include, but are not limited to, antibodies for cancercells, antibodies for viruses, antibodies that bind to cell surfacereceptors (for example, CD8, CD34, and CD45), and therapeuticantibodies.

As used herein the term “associated” or “associated with” can mean thattwo or more species are identifiable as being co-located at a point intime. An association can mean that two or more species are or werewithin a similar container. An association can be an informaticsassociation. For example, digital information regarding two or morespecies can be stored and can be used to determine that one or more ofthe species were co-located at a point in time. An association can alsobe a physical association. In some embodiments, two or more associatedspecies are “tethered”, “attached”, or “immobilized” to one another orto a common solid or semisolid surface. An association may refer tocovalent or non-covalent means for attaching labels to solid orsemi-solid supports such as beads. An association may be a covalent bondbetween a target and a label. An association can comprise hybridizationbetween two molecules (such as a target molecule and a label).

As used herein, the term “complementary” can refer to the capacity forprecise pairing between two nucleotides. For example, if a nucleotide ata given position of a nucleic acid is capable of hydrogen bonding with anucleotide of another nucleic acid, then the two nucleic acids areconsidered to be complementary to one another at that position.Complementarity between two single-stranded nucleic acid molecules maybe “partial,” in which only some of the nucleotides bind, or it may becomplete when total complementarity exists between the single-strandedmolecules. A first nucleotide sequence can be said to be the“complement” of a second sequence if the first nucleotide sequence iscomplementary to the second nucleotide sequence. A first nucleotidesequence can be said to be the “reverse complement” of a secondsequence, if the first nucleotide sequence is complementary to asequence that is the reverse (i.e., the order of the nucleotides isreversed) of the second sequence. As used herein, the terms“complement”, “complementary”, and “reverse complement” can be usedinterchangeably. It is understood from the disclosure that if a moleculecan hybridize to another molecule it may be the complement of themolecule that is hybridizing.

As used herein, the term “digital counting” can refer to a method forestimating a number of target molecules in a sample. Digital countingcan include the step of determining a number of unique labels that havebeen associated with targets in a sample. This methodology, which can bestochastic in nature, transforms the problem of counting molecules fromone of locating and identifying identical molecules to a series ofyes/no digital questions regarding detection of a set of predefinedlabels.

As used herein, the term “label” or “labels” can refer to nucleic acidcodes associated with a target within a sample. A label can be, forexample, a nucleic acid label. A label can be an entirely or partiallyamplifiable label. A label can be entirely or partially sequencablelabel. A label can be a portion of a native nucleic acid that isidentifiable as distinct. A label can be a known sequence. A label cancomprise a junction of nucleic acid sequences, for example a junction ofa native and non-native sequence. As used herein, the term “label” canbe used interchangeably with the terms, “index”, “tag,” or “label-tag.”Labels can convey information. For example, in various embodiments,labels can be used to determine an identity of a sample, a source of asample, an identity of a cell, and/or a target.

As used herein, the term “non-depleting reservoirs” can refer to a poolof barcodes (e.g., stochastic barcodes) made up of many differentlabels. A non-depleting reservoir can comprise large numbers ofdifferent barcodes such that when the non-depleting reservoir isassociated with a pool of targets each target is likely to be associatedwith a unique barcode. The uniqueness of each labeled target moleculecan be determined by the statistics of random choice, and depends on thenumber of copies of identical target molecules in the collectioncompared to the diversity of labels. The size of the resulting set oflabeled target molecules can be determined by the stochastic nature ofthe barcoding process, and analysis of the number of barcodes detectedthen allows calculation of the number of target molecules present in theoriginal collection or sample. When the ratio of the number of copies ofa target molecule present to the number of unique barcodes is low, thelabeled target molecules are highly unique (i.e., there is a very lowprobability that more than one target molecule will have been labeledwith a given label).

As used herein, the term “nucleic acid” refers to a polynucleotidesequence, or fragment thereof. A nucleic acid can comprise nucleotides.A nucleic acid can be exogenous or endogenous to a cell. A nucleic acidcan exist in a cell-free environment. A nucleic acid can be a gene orfragment thereof. A nucleic acid can be DNA. A nucleic acid can be RNA.A nucleic acid can comprise one or more analogs (e.g., altered backbone,sugar, or nucleobase). Some non-limiting examples of analogs include:5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos,locked nucleic acids, glycol nucleic acids, threose nucleic acids,dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g.,rhodamine or fluorescein linked to the sugar), thiol containingnucleotides, biotin linked nucleotides, fluorescent base analogs, CpGislands, methyl-7-guanosine, methylated nucleotides, inosine,thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.“Nucleic acid”, “polynucleotide, “target polynucleotide”, and “targetnucleic acid” can be used interchangeably.

A nucleic acid can comprise one or more modifications (e.g., a basemodification, a backbone modification), to provide the nucleic acid witha new or enhanced feature (e.g., improved stability). A nucleic acid cancomprise a nucleic acid affinity tag. A nucleoside can be a base-sugarcombination. The base portion of the nucleoside can be a heterocyclicbase. The two most common classes of such heterocyclic bases are thepurines and the pyrimidines. Nucleotides can be nucleosides that furtherinclude a phosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, the 3′, or the 5′ hydroxylmoiety of the sugar. In forming nucleic acids, the phosphate groups cancovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound;however, linear compounds are generally suitable. In addition, linearcompounds may have internal nucleotide base complementarity and maytherefore fold in a manner as to produce a fully or partiallydouble-stranded compound. Within nucleic acids, the phosphate groups cancommonly be referred to as forming the internucleoside backbone of thenucleic acid. The linkage or backbone can be a 3′ to 5′ phosphodiesterlinkage.

A nucleic acid can comprise a modified backbone and/or modifiedinternucleoside linkages. Modified backbones can include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. Suitable modified nucleic acidbackbones containing a phosphorus atom therein can include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonate such as 3′-alkylene phosphonates, 5′-alkylene phosphonates,chiral phosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates, and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs, and those havinginverted polarity wherein one or more internucleotide linkages is a 3′to 3′, a 5′ to 5′ or a 2′ to 2′ linkage.

A nucleic acid can comprise polynucleotide backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These can include those having morpholino linkages (formed in part fromthe sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; riboacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; and others having mixed N, O, Sand CH₂ component parts.

A nucleic acid can comprise a nucleic acid mimetic. The term “mimetic”can be intended to include polynucleotides wherein only the furanosering or both the furanose ring and the internucleotide linkage arereplaced with non-furanose groups, replacement of only the furanose ringcan also be referred as being a sugar surrogate. The heterocyclic basemoiety or a modified heterocyclic base moiety can be maintained forhybridization with an appropriate target nucleic acid. One such nucleicacid can be a peptide nucleic acid (PNA). In a PNA, the sugar-backboneof a polynucleotide can be replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleotides can beretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. The backbone in PNA compounds cancomprise two or more linked aminoethylglycine units which gives PNA anamide containing backbone. The heterocyclic base moieties can be bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone.

A nucleic acid can comprise a morpholino backbone structure. Forexample, a nucleic acid can comprise a 6-membered morpholino ring inplace of a ribose ring. In some of these embodiments, aphosphorodiamidate or other non-phosphodiester internucleoside linkagecan replace a phosphodiester linkage.

A nucleic acid can comprise linked morpholino units (e.g., morpholinonucleic acid) having heterocyclic bases attached to the morpholino ring.Linking groups can link the morpholino monomeric units in a morpholinonucleic acid. Non-ionic morpholino-based oligomeric compounds can haveless undesired interactions with cellular proteins. Morpholino-basedpolynucleotides can be nonionic mimics of nucleic acids. A variety ofcompounds within the morpholino class can be joined using differentlinking groups. A further class of polynucleotide mimetic can bereferred to as cyclohexenyl nucleic acids (CeNA). The furanose ringnormally present in a nucleic acid molecule can be replaced with acyclohexenyl ring. CeNA DMT protected phosphoramidite monomers can beprepared and used for oligomeric compound synthesis usingphosphoramidite chemistry. The incorporation of CeNA monomers into anucleic acid chain can increase the stability of a DNA/RNA hybrid. CeNAoligoadenylates can form complexes with nucleic acid complements withsimilar stability to the native complexes. A further modification caninclude Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group islinked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. Thelinkage can be a methylene (—CH₂), group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. LNA and LNA analogs can displayvery high duplex thermal stabilities with complementary nucleic acid(Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradation andgood solubility properties.

A nucleic acid may also include nucleobase (often referred to simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases can include the purine bases, (e.g., adenine (A)and guanine (G)), and the pyrimidine bases, (e.g., thymine (T), cytosine(C) and uracil (U)). Modified nucleobases can include other syntheticand natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C═C—CH3) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modifiednucleobases can include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one),G-clamps such as a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindolecytidine (H-pyrido(3′,2′:4,5)pyrrolo[2,3-d]pyrimidin-2-one).

As used herein, the term “sample” can refer to a composition comprisingtargets. Suitable samples for analysis by the disclosed methods,devices, and systems include cells, tissues, organs, or organisms.

As used herein, the term “sampling device” or “device” can refer to adevice which may take a section of a sample and/or place the section ona substrate. A sample device can refer to, for example, a fluorescenceactivated cell sorting (FACS) machine, a cell sorter machine, a biopsyneedle, a biopsy device, a tissue sectioning device, a microfluidicdevice, a blade grid, and/or a microtome.

As used herein, the term “solid support” can refer to discrete solid orsemi-solid surfaces to which a plurality of barcodes (e.g., stochasticbarcodes) may be attached. A solid support may encompass any type ofsolid, porous, or hollow sphere, ball, bearing, cylinder, or othersimilar configuration composed of plastic, ceramic, metal, or polymericmaterial (e.g., hydrogel) onto which a nucleic acid may be immobilized(e.g., covalently or non-covalently). A solid support may comprise adiscrete particle that may be spherical (e.g., microspheres) or have anon-spherical or irregular shape, such as cubic, cuboid, pyramidal,cylindrical, conical, oblong, or disc-shaped, and the like. A bead canbe non-spherical in shape. A plurality of solid supports spaced in anarray may not comprise a substrate. A solid support may be usedinterchangeably with the term “bead.”

As used herein, the term “stochastic barcode” can refer to apolynucleotide sequence comprising labels of the present disclosure. Astochastic barcode can be a polynucleotide sequence that can be used forstochastic barcoding. Stochastic barcodes can be used to quantifytargets within a sample. Stochastic barcodes can be used to control forerrors which may occur after a label is associated with a target. Forexample, a stochastic barcode can be used to assess amplification orsequencing errors. A stochastic barcode associated with a target can becalled a stochastic barcode-target or stochastic barcode-tag-target.

As used herein, the term “gene-specific stochastic barcode” can refer toa polynucleotide sequence comprising labels and a target-binding regionthat is gene-specific. A stochastic barcode can be a polynucleotidesequence that can be used for stochastic barcoding. Stochastic barcodescan be used to quantify targets within a sample. Stochastic barcodes canbe used to control for errors which may occur after a label isassociated with a target. For example, a stochastic barcode can be usedto assess amplification or sequencing errors. A stochastic barcodeassociated with a target can be called a stochastic barcode-target orstochastic barcode-tag-target.

As used herein, the term “stochastic barcoding” can refer to the randomlabeling (e.g., barcoding) of nucleic acids. Stochastic barcoding canutilize a recursive Poisson strategy to associate and quantify labelsassociated with targets. As used herein, the term “stochastic barcoding”can be used interchangeably with “stochastic labeling.”

As used here, the term “target” can refer to a composition which can beassociated with a barcode (e.g., a stochastic barcode). Exemplarysuitable targets for analysis by the disclosed methods, devices, andsystems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, andthe like. Targets can be single or double stranded. In some embodiments,targets can be proteins, peptides, or polypeptides. In some embodiments,targets are lipids. As used herein, “target” can be used interchangeablywith “species.”

As used herein, the term “reverse transcriptases” can refer to a groupof enzymes having reverse transcriptase activity (i.e., that catalyzesynthesis of DNA from an RNA template). In general, such enzymesinclude, but are not limited to, retroviral reverse transcriptase,retrotransposon reverse transcriptase, retroplasmid reversetranscriptases, retron reverse transcriptases, bacterial reversetranscriptases, group II intron-derived reverse transcriptase, andmutants, variants or derivatives thereof. Non-retroviral reversetranscriptases include non-LTR retrotransposon reverse transcriptases,retroplasmid reverse transcriptases, retron reverse transciptases, andgroup II intron reverse transcriptases. Examples of group II intronreverse transcriptases include the Lactococcus lactis LLLtrB intronreverse transcriptase, the Thermosynechococcus elongatus TeI4c intronreverse transcriptase, or the Geobacillus stearothermophilus GsI-IICintron reverse transcriptase. Other classes of reverse transcriptasescan include many classes of non-retroviral reverse transcriptases (i.e.,retrons, group II introns, and diversity-generating retroelements amongothers).

The terms “universal adaptor primer,” “universal primer adaptor” or“universal adaptor sequence” are used interchangeably to refer to anucleotide sequence that can be used to hybridize to barcodes (e.g.,stochastic barcodes) to generate gene-specific barcodes. A universaladaptor sequence can, for example, be a known sequence that is universalacross all barcodes used in methods of the disclosure. For example, whenmultiple targets are being labeled using the methods disclosed herein,each of the target-specific sequences may be linked to the sameuniversal adaptor sequence. In some embodiments, more than one universaladaptor sequences may be used in the methods disclosed herein. Forexample, when multiple targets are being labeled using the methodsdisclosed herein, at least two of the target-specific sequences arelinked to different universal adaptor sequences. A universal adaptorprimer and its complement may be included in two oligonucleotides, oneof which comprises a target-specific sequence and the other comprises abarcode. For example, a universal adaptor sequence may be part of anoligonucleotide comprising a target-specific sequence to generate anucleotide sequence that is complementary to a target nucleic acid. Asecond oligonucleotide comprising a barcode and a complementary sequenceof the universal adaptor sequence may hybridize with the nucleotidesequence and generate a target-specific barcode (e.g., a target-specificstochastic barcode). In some embodiments, a universal adaptor primer hasa sequence that is different from a universal PCR primer used in themethods of this disclosure.

Barcodes

Barcoding, such as stochastic barcoding, has been described in, forexample, US20150299784, WO2015031691, and Fu et al, Proc Natl Acad SciU.S.A. 2011 May 31; 108(22):9026-31, the content of these publicationsis incorporated hereby in its entirety. In some embodiments, the barcodedisclosed herein can be a stochastic barcode which can be apolynucleotide sequence that may be used to stochastically label (e.g.,barcode, tag) a target. Barcodes can be referred to stochastic barcodesif the ratio of the number of different barcode sequences of thestochastic barcodes and the number of occurrence of any of the targetsto be labeled can be, or be about, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or arange between any two of these values. A target can be an mRNA speciescomprising mRNA molecules with identical or nearly identical sequences.Barcodes can be referred to as stochastic barcodes if the ratio of thenumber of different barcode sequences of the stochastic barcodes and thenumber of occurrence of any of the targets to be labeled is at least, oris at most, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1,60:1, 70:1, 80:1, 90:1, or 100:1. Barcode sequences of stochasticbarcodes can be referred to as molecular labels.

A barcode, for example a stochastic barcode, can comprise one or morelabels. Exemplary labels can include a universal label, a cell label, abarcode sequence (e.g., a molecular label), a sample label, a platelabel, a spatial label, and/or a pre-spatial label. FIG. 1 illustratesan exemplary barcode 104 with a spatial label. The barcode 104 cancomprise a 5′ amine that may link the barcode to a solid support 105.The barcode can comprise a universal label, a dimension label, a spatiallabel, a cell label, and/or a molecular label. The order of differentlabels (including but not limited to the universal label, the dimensionlabel, the spatial label, the cell label, and the molecule label) in thebarcode can vary. For example, as shown in FIG. 1, the universal labelmay be the 5′-most label, and the molecular label may be the 3′-mostlabel. The spatial label, dimension label, and the cell label may be inany order. In some embodiments, the universal label, the spatial label,the dimension label, the cell label, and the molecular label are in anyorder. The barcode can comprise a target-binding region. Thetarget-binding region can interact with a target (e.g., target nucleicacid, RNA, mRNA, DNA) in a sample. For example, a target-binding regioncan comprise an oligo(dT) sequence which can interact with poly(A) tailsof mRNAs. In some instances, the labels of the barcode (e.g., universallabel, dimension label, spatial label, cell label, and barcode sequence)may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 or more nucleotides.

A label, for example the cell label, can comprise a unique set ofnucleic acid sub-sequences of defined length, e.g., seven nucleotideseach (equivalent to the number of bits used in some Hamming errorcorrection codes), which can be designed to provide error correctioncapability. The set of error correction sub-sequences comprise sevennucleotide sequences can be designed such that any pairwise combinationof sequences in the set exhibits a defined “genetic distance” (or numberof mismatched bases), for example, a set of error correctionsub-sequences can be designed to exhibit a genetic distance of threenucleotides. In this case, review of the error correction sequences inthe set of sequence data for labeled target nucleic acid molecules(described more fully below) can allow one to detect or correctamplification or sequencing errors. In some embodiments, the length ofthe nucleic acid sub-sequences used for creating error correction codescan vary, for example, they can be, or be about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 31, 40, 50, or a number or a range between any two ofthese values, nucleotides in length. In some embodiments, nucleic acidsub-sequences of other lengths can be used for creating error correctioncodes.

The barcode can comprise a target-binding region. The target-bindingregion can interact with a target in a sample. The target can be, orcomprise, ribonucleic acids (RNAs), messenger RNAs (mRNAs), microRNAs,small interfering RNAs (siRNAs), RNA degradation products, RNAs eachcomprising a poly(A) tail, or any combination thereof. In someembodiments, the plurality of targets can include deoxyribonucleic acids(DNAs).

In some embodiments, a target-binding region can comprise an oligo(dT)sequence which can interact with poly(A) tails of mRNAs. One or more ofthe labels of the barcode (e.g., the universal label, the dimensionlabel, the spatial label, the cell label, and the barcode sequences(e.g., molecular label)) can be separated by a spacer from another oneor two of the remaining labels of the barcode. The spacer can be, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, or more nucleotides. In some embodiments, none of the labelsof the barcode is separated by spacer.

Universal Labels

A barcode can comprise one or more universal labels. In someembodiments, the one or more universal labels can be the same for allbarcodes in the set of barcodes attached to a given solid support. Insome embodiments, the one or more universal labels can be the same forall barcodes attached to a plurality of beads. In some embodiments, auniversal label can comprise a nucleic acid sequence that is capable ofhybridizing to a sequencing primer. Sequencing primers can be used forsequencing barcodes comprising a universal label. Sequencing primers(e.g., universal sequencing primers) can comprise sequencing primersassociated with high-throughput sequencing platforms. In someembodiments, a universal label can comprise a nucleic acid sequence thatis capable of hybridizing to a PCR primer. In some embodiments, theuniversal label can comprise a nucleic acid sequence that is capable ofhybridizing to a sequencing primer and a PCR primer. The nucleic acidsequence of the universal label that is capable of hybridizing to asequencing or PCR primer can be referred to as a primer binding site. Auniversal label can comprise a sequence that can be used to initiatetranscription of the barcode. A universal label can comprise a sequencethat can be used for extension of the barcode or a region within thebarcode. A universal label can be, or be about, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, or a number or a range between any two ofthese values, nucleotides in length. For example, a universal label cancomprise at least about 10 nucleotides. A universal label can be atleast, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,100, 200, or 300 nucleotides in length. In some embodiments, a cleavablelinker or modified nucleotide can be part of the universal labelsequence to enable the barcode to be cleaved off from the support.

Dimension Labels

A barcode can comprise one or more dimension labels. In someembodiments, a dimension label can comprise a nucleic acid sequence thatprovides information about a dimension in which the labeling (e.g.,stochastic labeling) occurred. For example, a dimension label canprovide information about the time at which a target was barcoded. Adimension label can be associated with a time of barcoding (e.g.,stochastic barcoding) in a sample. A dimension label can be activated atthe time of labeling. Different dimension labels can be activated atdifferent times. The dimension label provides information about theorder in which targets, groups of targets, and/or samples were barcoded.For example, a population of cells can be barcoded at the G0 phase ofthe cell cycle. The cells can be pulsed again with barcodes (e.g.,stochastic barcodes) at the G1 phase of the cell cycle. The cells can bepulsed again with barcodes at the S phase of the cell cycle, and so on.Barcodes at each pulse (e.g., each phase of the cell cycle), cancomprise different dimension labels. In this way, the dimension labelprovides information about which targets were labelled at which phase ofthe cell cycle. Dimension labels can interrogate many differentbiological times. Exemplary biological times can include, but are notlimited to, the cell cycle, transcription (e.g., transcriptioninitiation), and transcript degradation. In another example, a sample(e.g., a cell, a population of cells) can be labeled before and/or aftertreatment with a drug and/or therapy. The changes in the number ofcopies of distinct targets can be indicative of the sample's response tothe drug and/or therapy.

A dimension label can be activatable. An activatable dimension label canbe activated at a specific time point. The activatable label can be, forexample, constitutively activated (e.g., not turned off). Theactivatable dimension label can be, for example, reversibly activated(e.g., the activatable dimension label can be turned on and turned off).The dimension label can be, for example, reversibly activatable at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The dimension label can bereversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more times. In some embodiments, the dimension label can beactivated with fluorescence, light, a chemical event (e.g., cleavage,ligation of another molecule, addition of modifications (e.g.,pegylated, sumoylated, acetylated, methylated, deacetylated,demethylated), a photochemical event (e.g., photocaging), andintroduction of a non-natural nucleotide.

The dimension label can, in some embodiments, be identical for allbarcodes (e.g., stochastic barcodes) attached to a given solid support(e.g., a bead), but different for different solid supports (e.g.,beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%,99% or 100%, of barcodes on the same solid support can comprise the samedimension label. In some embodiments, at least 60% of barcodes on thesame solid support can comprise the same dimension label. In someembodiments, at least 95% of barcodes on the same solid support cancomprise the same dimension label.

There can be as many as 10⁶ or more unique dimension label sequencesrepresented in a plurality of solid supports (e.g., beads). A dimensionlabel can be, or be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, or a number or a range between any two of these values, nucleotidesin length. A dimension label can be at least, or be at most, 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300, nucleotides inlength. A dimension label can comprise between about 5 to about 200nucleotides. A dimension label can comprise between about 10 to about150 nucleotides. A dimension label can comprise between about 20 toabout 125 nucleotides in length.

Spatial Labels

A barcode can comprise one or more spatial labels. In some embodiments,a spatial label can comprise a nucleic acid sequence that providesinformation about the spatial orientation of a target molecule which isassociated with the barcode. A spatial label can be associated with acoordinate in a sample. The coordinate can be a fixed coordinate. Forexample, a coordinate can be fixed in reference to a substrate. Aspatial label can be in reference to a two or three-dimensional grid. Acoordinate can be fixed in reference to a landmark. The landmark can beidentifiable in space. A landmark can be a structure which can beimaged. A landmark can be a biological structure, for example ananatomical landmark. A landmark can be a cellular landmark, for instancean organelle. A landmark can be a non-natural landmark such as astructure with an identifiable identifier such as a color code, barcode, magnetic property, fluorescents, radioactivity, or a unique sizeor shape. A spatial label can be associated with a physical partition(e.g., A well, a container, or a droplet). In some embodiments, multiplespatial labels are used together to encode one or more positions inspace.

The spatial label can be identical for all barcodes attached to a givensolid support (e.g., a bead), but different for different solid supports(e.g., beads). In some embodiments, the percentage of barcodes on thesame solid support comprising the same spatial label can be, or beabout, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or arange between any two of these values. In some embodiments, thepercentage of barcodes on the same solid support comprising the samespatial label can be at least, or be at most, 60%, 70%, 80%, 85%, 90%,95%, 97%, 99%, or 100%. In some embodiments, at least 60% of barcodes onthe same solid support can comprise the same spatial label. In someembodiments, at least 95% of barcodes on the same solid support cancomprise the same spatial label.

There can be as many as 10⁶ or more unique spatial label sequencesrepresented in a plurality of solid supports (e.g., beads). A spatiallabel can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, or a number or a range between any two of these values,nucleotides in length. A spatial label can be at least or at most 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300nucleotides in length. A spatial label can comprise between about 5 toabout 200 nucleotides. A spatial label can comprise between about 10 toabout 150 nucleotides. A spatial label can comprise between about 20 toabout 125 nucleotides in length.

Cell Labels

A barcode (e.g., a stochastic barcode) can comprise one or more celllabels. In some embodiments, a cell label can comprise a nucleic acidsequence that provides information for determining which target nucleicacid originated from which cell. In some embodiments, the cell label isidentical for all barcodes attached to a given solid support (e.g., abead), but different for different solid supports (e.g., beads). In someembodiments, the percentage of barcodes on the same solid supportcomprising the same cell label can be, or be about 60%, 70%, 80%, 85%,90%, 95%, 97%, 99%, 100%, or a number or a range between any two ofthese values. In some embodiments, the percentage of barcodes on thesame solid support comprising the same cell label can be, or be about60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. For example, at least60% of barcodes on the same solid support can comprise the same celllabel. As another example, at least 95% of barcodes on the same solidsupport can comprise the same cell label.

There can be as many as 10⁶ or more unique cell label sequencesrepresented in a plurality of solid supports (e.g., beads). A cell labelcan be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,or a number or a range between any two of these values, nucleotides inlength. A cell label can be at least, or be at most, 1, 2, 3, 4, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.For example, a cell label can comprise between about 5 to about 200nucleotides. As another example, a cell label can comprise between about10 to about 150 nucleotides. As yet another example, a cell label cancomprise between about 20 to about 125 nucleotides in length.

Barcode Sequences

A barcode can comprise one or more barcode sequences. In someembodiments, a barcode sequence can comprise a nucleic acid sequencethat provides identifying information for the specific type of targetnucleic acid species hybridized to the barcode. A barcode sequence cancomprise a nucleic acid sequence that provides a counter (e.g., thatprovides a rough approximation) for the specific occurrence of thetarget nucleic acid species hybridized to the barcode (e.g.,target-binding region).

In some embodiments, a diverse set of barcode sequences are attached toa given solid support (e.g., a bead). In some embodiments, there can be,or be about, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or a number or arange between any two of these values, unique molecular label sequences.For example, a plurality of barcodes can comprise about 6561 barcodessequences with distinct sequences. As another example, a plurality ofbarcodes can comprise about 65536 barcode sequences with distinctsequences. In some embodiments, there can be at least, or be at most,10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹, unique barcode sequences. Theunique molecular label sequences can be attached to a given solidsupport (e.g., a bead).

The length of a barcode can be different in different implementations.For example, a barcode can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, or a number or a range between any two of thesevalues, nucleotides in length. As another example, a barcode can be atleast, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,100, 200, or 300 nucleotides in length.

Molecular Labels

A barcode (e.g., a stochastic barcode) can comprise one or moremolecular labels. Molecular labels can include barcode sequences. Insome embodiments, a molecular label can comprise a nucleic acid sequencethat provides identifying information for the specific type of targetnucleic acid species hybridized to the barcode. A molecular label cancomprise a nucleic acid sequence that provides a counter for thespecific occurrence of the target nucleic acid species hybridized to thebarcode (e.g., target-binding region).

In some embodiments, a diverse set of molecular labels are attached to agiven solid support (e.g., a bead). In some embodiments, there can be,or be about, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or a number or arange between any two of these values, of unique molecular labelsequences. For example, a plurality of barcodes can comprise about 6561molecular labels with distinct sequences. As another example, aplurality of barcodes can comprise about 65536 molecular labels withdistinct sequences. In some embodiments, there can be at least, or be atmost, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹, unique molecular labelsequences. Barcodes with unique molecular label sequences can beattached to a given solid support (e.g., a bead).

For stochastic barcoding using a plurality of stochastic barcodes, theratio of the number of different molecular label sequences and thenumber of occurrence of any of the targets can be, or be about, 1:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1,15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1,90:1, 100:1, or a number or a range between any two of these values. Atarget can be an mRNA species comprising mRNA molecules with identicalor nearly identical sequences. In some embodiments, the ratio of thenumber of different molecular label sequences and the number ofoccurrence of any of the targets is at least, or is at most, 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1,or 100:1.

A molecular label can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, or a number or a range between any two of thesevalues, nucleotides in length. A molecular label can be at least, or beat most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or300 nucleotides in length.

Target-Binding Region

A barcode can comprise one or more target binding regions, such ascapture probes. In some embodiments, a target-binding region canhybridize with a target of interest. In some embodiments, the targetbinding regions can comprise a nucleic acid sequence that hybridizesspecifically to a target (e.g., target nucleic acid, target molecule,e.g., a cellular nucleic acid to be analyzed), for example to a specificgene sequence. In some embodiments, a target binding region can comprisea nucleic acid sequence that can attach (e.g., hybridize) to a specificlocation of a specific target nucleic acid. In some embodiments, thetarget binding region can comprise a nucleic acid sequence that iscapable of specific hybridization to a restriction enzyme site overhang(e.g., an EcoRI sticky-end overhang). The barcode can then ligate to anynucleic acid molecule comprising a sequence complementary to therestriction site overhang.

In some embodiments, a target binding region can comprise a non-specifictarget nucleic acid sequence. A non-specific target nucleic acidsequence can refer to a sequence that can bind to multiple targetnucleic acids, independent of the specific sequence of the targetnucleic acid. For example, target binding region can comprise a randommultimer sequence, or an oligo(dT) sequence that hybridizes to thepoly(A) tail on mRNA molecules. A random multimer sequence can be, forexample, a random dimer, trimer, quatramer, pentamer, hexamer, septamer,octamer, nonamer, decamer, or higher multimer sequence of any length. Insome embodiments, the target binding region is the same for all barcodesattached to a given bead. In some embodiments, the target bindingregions for the plurality of barcodes attached to a given bead cancomprise two or more different target binding sequences. A targetbinding region can be, or be about, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, or a number or a range between any two of these values, nucleotidesin length. A target binding region can be at most about 5, 10, 15, 20,25, 30, 35, 40, 45, 50 or more nucleotides in length.

In some embodiments, a target-binding region can comprise an oligo(dT)which can hybridize with mRNAs comprising polyadenylated ends. Atarget-binding region can be gene-specific. For example, atarget-binding region can be configured to hybridize to a specificregion of a target. A target-binding region can be, or be about, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26 27, 28, 29, 30, or a number or a range between any two ofthese values, nucleotides in length. A target-binding region can be atleast, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30,nucleotides in length. A target-binding region can be about 5-30nucleotides in length. When a barcode comprises a gene-specifictarget-binding region, the barcode can be referred to herein as agene-specific barcode.

Orientation Property

A stochastic barcode (e.g., a stochastic barcode) can comprise one ormore orientation properties which can be used to orient (e.g., align)the barcodes. A barcode can comprise a moiety for isoelectric focusing.Different barcodes can comprise different isoelectric focusing points.When these barcodes are introduced to a sample, the sample can undergoisoelectric focusing in order to orient the barcodes into a known way.In this way, the orientation property can be used to develop a known mapof barcodes in a sample. Exemplary orientation properties can include,electrophoretic mobility (e.g., based on size of the barcode),isoelectric point, spin, conductivity, and/or self-assembly. Forexample, barcodes with an orientation property of self-assembly, canself-assemble into a specific orientation (e.g., nucleic acidnanostructure) upon activation.

Affinity Property

A barcode (e.g., a stochastic barcode) can comprise one or more affinityproperties. For example, a spatial label can comprise an affinityproperty. An affinity property can include a chemical and/or biologicalmoiety that can facilitate binding of the barcode to another entity(e.g., cell receptor). For example, an affinity property can comprise anantibody, for example, an antibody specific for a specific moiety (e.g.,receptor) on a sample. In some embodiments, the antibody can guide thebarcode to a specific cell type or molecule. Targets at and/or near thespecific cell type or molecule can be labeled (e.g., stochasticallylabeled). The affinity property can, in some embodiments, providespatial information in addition to the nucleotide sequence of thespatial label because the antibody can guide the barcode to a specificlocation. The antibody can be a therapeutic antibody, for example amonoclonal antibody or a polyclonal antibody. The antibody can behumanized or chimeric. The antibody can be a naked antibody or a fusionantibody.

The antibody can be a full-length (i.e., naturally occurring or formedby normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive (i.e., specifically binding) portion of an immunoglobulinmolecule, like an antibody fragment.

The antibody fragment can be, for example, a portion of an antibody suchas F(ab′)2, Fab′, Fab, Fv, sFv and the like. In some embodiments, theantibody fragment can bind with the same antigen that is recognized bythe full-length antibody. The antibody fragment can include isolatedfragments consisting of the variable regions of antibodies, such as the“Fv” fragments consisting of the variable regions of the heavy and lightchains and recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”). Exemplary antibodies can include, but are not limited to,antibodies for cancer cells, antibodies for viruses, antibodies thatbind to cell surface receptors (CD8, CD34, CD45), and therapeuticantibodies.

Universal Adaptor Primer

A barcode can comprise one or more universal adaptor primers. Forexample, a gene-specific barcode, such as a gene-specific stochasticbarcode, can comprise a universal adaptor primer. A universal adaptorprimer can refer to a nucleotide sequence that is universal across allbarcodes. A universal adaptor primer can be used for buildinggene-specific barcodes. A universal adaptor primer can be, or be about,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26 27, 28, 29, 30, or a number or a range betweenany two of these nucleotides in length. A universal adaptor primer canbe at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30nucleotides in length. A universal adaptor primer can be from 5-30nucleotides in length.

Linker

When a barcode comprises more than one of a type of label (e.g., morethan one cell label or more than one barcode sequence, such as onemolecular label), the labels may be interspersed with a linker labelsequence. A linker label sequence can be at least about 5, 10, 15, 20,25, 30, 35, 40, 45, 50 or more nucleotides in length. A linker labelsequence can be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 ormore nucleotides in length. In some instances, a linker label sequenceis 12 nucleotides in length. A linker label sequence can be used tofacilitate the synthesis of the barcode. The linker label can comprisean error-correcting (e.g., Hamming) code.

Solid Supports

Barcodes, such as stochastic barcodes, disclosed herein can, in someembodiments, be associated with a solid support. The solid support canbe, for example, a synthetic particle. In some embodiments, some or allof the barcode sequences, such as molecular labels for stochasticbarcodes (e.g., the first barcode sequences) of a plurality of barcodes(e.g., the first plurality of barcodes) on a solid support differ by atleast one nucleotide. The cell labels of the barcodes on the same solidsupport can be the same. The cell labels of the barcodes on differentsolid supports can differ by at least one nucleotide. For example, firstcell labels of a first plurality of barcodes on a first solid supportcan have the same sequence, and second cell labels of a second pluralityof barcodes on a second solid support can have the same sequence. Thefirst cell labels of the first plurality of barcodes on the first solidsupport and the second cell labels of the second plurality of barcodeson the second solid support can differ by at least one nucleotide. Acell label can be, for example, about 5-20 nucleotides long. A barcodesequence can be, for example, about 5-20 nucleotides long. The syntheticparticle can be, for example, a bead.

The bead can be, for example, a silica gel bead, a controlled pore glassbead, a magnetic bead, a Dynabead, a Sephadex/Sepharose bead, acellulose bead, a polystyrene bead, or any combination thereof. The beadcan comprise a material such as polydimethylsiloxane (PDMS),polystyrene, glass, polypropylene, agarose, gelatin, hydrogel,paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer,titanium, latex, Sepharose, cellulose, nylon, silicone, or anycombination thereof.

In some embodiments, the bead can be a polymeric bead, for example adeformable bead or a gel bead, functionalized with barcodes orstochastic barcodes (such as gel beads from 10× Genomics (San Francisco,Calif.). In some implementation, a gel bead can comprise a polymer basedgels. Gel beads can be generated, for example, by encapsulating one ormore polymeric precursors into droplets. Upon exposure of the polymericprecursors to an accelerator (e.g., tetramethylethylenediamine (TEMED)),a gel bead may be generated.

In some embodiments, the particle can be degradable. For example, thepolymeric bead can dissolve, melt, or degrade, for example, under adesired condition. The desired condition can include an environmentalcondition. The desired condition may result in the polymeric beaddissolving, melting, or degrading in a controlled manner. A gel bead maydissolve, melt, or degrade due to a chemical stimulus, a physicalstimulus, a biological stimulus, a thermal stimulus, a magneticstimulus, an electric stimulus, a light stimulus, or any combinationthereof.

Analytes and/or reagents, such as oligonucleotide barcodes, for example,may be coupled/immobilized to the interior surface of a gel bead (e.g.,the interior accessible via diffusion of an oligonucleotide barcodeand/or materials used to generate an oligonucleotide barcode) and/or theouter surface of a gel bead or any other microcapsule described herein.Coupling/immobilization may be via any form of chemical bonding (e.g.,covalent bond, ionic bond) or physical phenomena (e.g., Van der Waalsforces, dipole-dipole interactions, etc.). In some embodiments,coupling/immobilization of a reagent to a gel bead or any othermicrocapsule described herein may be reversible, such as, for example,via a labile moiety (e.g., via a chemical cross-linker, includingchemical cross-linkers described herein). Upon application of astimulus, the labile moiety may be cleaved and the immobilized reagentset free. In some embodiments, the labile moiety is a disulfide bond.For example, in the case where an oligonucleotide barcode is immobilizedto a gel bead via a disulfide bond, exposure of the disulfide bond to areducing agent can cleave the disulfide bond and free theoligonucleotide barcode from the bead. The labile moiety may be includedas part of a gel bead or microcapsule, as part of a chemical linker thatlinks a reagent or analyte to a gel bead or microcapsule, and/or as partof a reagent or analyte. In some embodiments, at least one barcode ofthe plurality of barcodes can be immobilized on the particle, partiallyimmobilized on the particle, enclosed in the particle, partiallyenclosed in the particle, or any combination thereof.

In some embodiments, a gel bead can comprise a wide range of differentpolymers including but not limited to: polymers, heat sensitivepolymers, photosensitive polymers, magnetic polymers, pH sensitivepolymers, salt-sensitive polymers, chemically sensitive polymers,polyelectrolytes, polysaccharides, peptides, proteins, and/or plastics.Polymers may include but are not limited to materials such aspoly(N-isopropylacrylamide) (PNIPAAm), poly(styrene sulfonate) (PSS),poly(allyl amine) (PAAm), poly(acrylic acid) (PAA), poly(ethylene imine)(PEI), poly(diallyldimethyl-ammonium chloride) (PDADMAC), poly(pyrolle)(PPy), poly(vinylpyrrolidone) (PVPON), poly(vinyl pyridine) (PVP),poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA),polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthaladehyde)(PTHF), poly(hexyl viologen) (PHV), poly(L-lysine) (PLL),poly(L-arginine) (PARG), poly(lactic-co-glycolic acid) (PLGA).

Numerous chemical stimuli can be used to trigger the disruption,dissolution, or degradation of the beads. Examples of these chemicalchanges may include, but are not limited to pH-mediated changes to thebead wall, disintegration of the bead wall via chemical cleavage ofcrosslink bonds, triggered depolymerization of the bead wall, and beadwall switching reactions. Bulk changes may also be used to triggerdisruption of the beads.

Bulk or physical changes to the microcapsule through various stimulialso offer many advantages in designing capsules to release reagents.Bulk or physical changes occur on a macroscopic scale, in which beadrupture is the result of mechano-physical forces induced by a stimulus.These processes may include, but are not limited to pressure inducedrupture, bead wall melting, or changes in the porosity of the bead wall.

Biological stimuli may also be used to trigger disruption, dissolution,or degradation of beads. Generally, biological triggers resemblechemical triggers, but many examples use biomolecules, or moleculescommonly found in living systems such as enzymes, peptides, saccharides,fatty acids, nucleic acids and the like. For example, beads may comprisepolymers with peptide cross-links that are sensitive to cleavage byspecific proteases. More specifically, one example may comprise amicrocapsule comprising GFLGK peptide cross links. Upon addition of abiological trigger such as the protease Cathepsin B, the peptide crosslinks of the shell well are cleaved and the contents of the beads arereleased. In other cases, the proteases may be heat-activated. Inanother example, beads comprise a shell wall comprising cellulose.Addition of the hydrolytic enzyme chitosan serves as biologic triggerfor cleavage of cellulosic bonds, depolymerization of the shell wall,and release of its inner contents.

The beads may also be induced to release their contents upon theapplication of a thermal stimulus. A change in temperature can cause avariety changes to the beads. A change in heat may cause melting of abead such that the bead wall disintegrates. In other cases, the heat mayincrease the internal pressure of the inner components of the bead suchthat the bead ruptures or explodes. In still other cases, the heat maytransform the bead into a shrunken dehydrated state. The heat may alsoact upon heat-sensitive polymers within the wall of a bead to causedisruption of the bead.

Inclusion of magnetic nanoparticles to the bead wall of microcapsulesmay allow triggered rupture of the beads as well as guide the beads inan array. A device of this disclosure may comprise magnetic beads foreither purpose. In one example, incorporation of Fe₃O₄ nanoparticlesinto polyelectrolyte containing beads triggers rupture in the presenceof an oscillating magnetic field stimulus.

A bead may also be disrupted, dissolved, or degraded as the result ofelectrical stimulation. Similar to magnetic particles described in theprevious section, electrically sensitive beads can allow for bothtriggered rupture of the beads as well as other functions such asalignment in an electric field, electrical conductivity or redoxreactions. In one example, beads containing electrically sensitivematerial are aligned in an electric field such that release of innerreagents can be controlled. In other examples, electrical fields mayinduce redox reactions within the bead wall itself that may increaseporosity.

A light stimulus may also be used to disrupt the beads. Numerous lighttriggers are possible and may include systems that use various moleculessuch as nanoparticles and chromophores capable of absorbing photons ofspecific ranges of wavelengths. For example, metal oxide coatings can beused as capsule triggers. UV irradiation of polyelectrolyte capsulescoated with SiO₂ may result in disintegration of the bead wall. In yetanother example, photo switchable materials such as azobenzene groupsmay be incorporated in the bead wall. Upon the application of UV orvisible light, chemicals such as these undergo a reversible cis-to-transisomerization upon absorption of photons. In this aspect, incorporationof photon switches result in a bead wall that may disintegrate or becomemore porous upon the application of a light trigger.

For example, in a non-limiting example of barcoding (e.g., stochasticbarcoding) illustrated in FIG. 2, after introducing cells such as singlecells onto a plurality of microwells of a microwell array at block 208,beads can be introduced onto the plurality of microwells of themicrowell array at block 212. Each microwell can comprise one bead. Thebeads can comprise a plurality of barcodes. A barcode can comprise a 5′amine region attached to a bead. The barcode can comprise a universallabel, a barcode sequence (e.g., a molecular label), a target-bindingregion, or any combination thereof.

The barcodes disclosed herein can be associated with (e.g., attached to)a solid support (e.g., a bead). The barcodes associated with a solidsupport can each comprise a barcode sequence selected from a groupcomprising at least 100 or 1000 barcode sequences with unique sequences.In some embodiments, different barcodes associated with a solid supportcan comprise barcode with different sequences. In some embodiments, apercentage of barcodes associated with a solid support comprises thesame cell label. For example, the percentage can be, or be about 60%,70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or a range betweenany two of these values. As another example, the percentage can be atleast, or be at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. Insome embodiments, barcodes associated with a solid support can have thesame cell label. The barcodes associated with different solid supportscan have different cell labels selected from a group comprising at least100 or 1000 cell labels with unique sequences.

The barcodes disclosed herein can be associated to (e.g., attached to) asolid support (e.g., a bead). In some embodiments, barcoding theplurality of targets in the sample can be performed with a solid supportincluding a plurality of synthetic particles associated with theplurality of barcodes. In some embodiments, the solid support caninclude a plurality of synthetic particles associated with the pluralityof barcodes. The spatial labels of the plurality of barcodes ondifferent solid supports can differ by at least one nucleotide. Thesolid support can, for example, include the plurality of barcodes in twodimensions or three dimensions. The synthetic particles can be beads.The beads can be silica gel beads, controlled pore glass beads, magneticbeads, Dynabeads, Sephadex/Sepharose beads, cellulose beads, polystyrenebeads, or any combination thereof. The solid support can include apolymer, a matrix, a hydrogel, a needle array device, an antibody, orany combination thereof. In some embodiments, the solid supports can befree floating. In some embodiments, the solid supports can be embeddedin a semi-solid or solid array. The barcodes may not be associated withsolid supports. The barcodes can be individual nucleotides. The barcodescan be associated with a substrate.

As used herein, the terms “tethered,” “attached,” and “immobilized,” areused interchangeably, and can refer to covalent or non-covalent meansfor attaching barcodes to a solid support. Any of a variety of differentsolid supports can be used as solid supports for attachingpre-synthesized barcodes or for in situ solid-phase synthesis ofbarcode.

In some embodiments, the solid support is a bead. The bead can compriseone or more types of solid, porous, or hollow sphere, ball, bearing,cylinder, or other similar configuration which a nucleic acid can beimmobilized (e.g., covalently or non-covalently). The bead can be, forexample, composed of plastic, ceramic, metal, polymeric material, or anycombination thereof. A bead can be, or comprise, a discrete particlethat is spherical (e.g., microspheres) or have a non-spherical orirregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical,oblong, or disc-shaped, and the like. In some embodiments, a bead can benon-spherical in shape.

Beads can comprise a variety of materials including, but not limited to,paramagnetic materials (e.g., magnesium, molybdenum, lithium, andtantalum), superparamagnetic materials (e.g., ferrite (Fe₃O₄; magnetite)nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt,some alloys thereof, and some rare earth metal compounds), ceramic,plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers,titanium, latex, Sepharose, agarose, hydrogel, polymer, cellulose,nylon, or any combination thereof.

In some embodiments, the bead (e.g., the bead to which the labels areattached) is a hydrogel bead. In some embodiments, the bead compriseshydrogel.

Some embodiments disclosed herein include one or more particles (forexample, beads). Each of the particles can comprise a plurality ofoligonucleotides (e.g., barcodes). Each of the plurality ofoligonucleotides can comprise a barcode sequence (e.g., a molecularlabel sequence), a cell label, and a target-binding region (e.g., anoligo(dT) sequence, a gene-specific sequence, a random multimer, or acombination thereof). The cell label sequence of each of the pluralityof oligonucleotides can be the same. The cell label sequences ofoligonucleotides on different particles can be different such that theoligonucleotides on different particles can be identified. The number ofdifferent cell label sequences can be different in differentimplementations. In some embodiments, the number of cell label sequencescan be, or be about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸,10⁹, a number or a range between any two of these values, or more. Insome embodiments, the number of cell label sequences can be at least, orbe at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000,50000, 60000, 70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸, or 10⁹. Insome embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, or more of the plurality of the particles include oligonucleotideswith the same cell sequence. In some embodiment, the plurality ofparticles that include oligonucleotides with the same cell sequence canbe at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more. In some embodiments, none ofthe plurality of the particles has the same cell label sequence.

The plurality of oligonucleotides on each particle can comprisedifferent barcode sequences (e.g., molecular labels). In someembodiments, the number of barcode sequences can be, or be about 10,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000,70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸, 10⁹, or a number or a rangebetween any two of these values. In some embodiments, the number ofbarcode sequences can be at least, or be at most 10, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000,100000, 10⁶, 10⁷, 10⁸, or 10⁹. For example, at least 100 of theplurality of oligonucleotides comprise different barcode sequences. Asanother example, in a single particle, at least 100, 500, 1000, 5000,10000, 15000, 20000, 50000, a number or a range between any two of thesevalues, or more of the plurality of oligonucleotides comprise differentbarcode sequences. Some embodiments provide a plurality of the particlescomprising barcodes. In some embodiments, the ratio of an occurrence (ora copy or a number) of a target to be labeled and the different barcodesequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30,1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or more. In some embodiments, eachof the plurality of oligonucleotides further comprises a sample label, auniversal label, or both. The particle can be, for example, ananoparticle or microparticle.

The size of the beads can vary. For example, the diameter of the beadcan range from 0.1 micrometer to 50 micrometer. In some embodiments, thediameter of the bead can be, or be about, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50 micrometer, or a number or a range between anytwo of these values.

The diameter of the bead can be related to the diameter of the wells ofthe substrate. In some embodiments, the diameter of the bead can be, orbe about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a numberor a range between any two of these values, longer or shorter than thediameter of the well. The diameter of the beads can be related to thediameter of a cell (e.g., a single cell entrapped by a well of thesubstrate). In some embodiments, the diameter of the bead can be atleast, or be at most, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% longer or shorter than the diameter of the well. The diameter ofthe beads can be related to the diameter of a cell (e.g., a single cellentrapped by a well of the substrate). In some embodiments, the diameterof the bead can be, or be about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 150%, 200%, 250%, 300%, or a number or a range between anytwo of these values, longer or shorter than the diameter of the cell. Insome embodiments, the diameter of the beads can be at least, or be atmost, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,250%, or 300% longer or shorter than the diameter of the cell.

A bead can be attached to and/or embedded in a substrate. A bead can beattached to and/or embedded in a gel, hydrogel, polymer and/or matrix.The spatial position of a bead within a substrate (e.g., gel, matrix,scaffold, or polymer) can be identified using the spatial label presenton the barcode on the bead which can serve as a location address.

Examples of beads can include, but are not limited to, streptavidinbeads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads,antibody conjugated beads (e.g., anti-immunoglobulin microbeads),protein A conjugated beads, protein G conjugated beads, protein A/Gconjugated beads, protein L conjugated beads, oligo(dT) conjugatedbeads, silica beads, silica-like beads, anti-biotin microbeads,anti-fluorochrome microbeads, and BcMag™ Carboxyl-Terminated MagneticBeads.

A bead can be associated with (e.g., impregnated with) quantum dots orfluorescent dyes to make it fluorescent in one fluorescence opticalchannel or multiple optical channels. A bead can be associated with ironoxide or chromium oxide to make it paramagnetic or ferromagnetic. Beadscan be identifiable. For example, a bead can be imaged using a camera. Abead can have a detectable code associated with the bead. For example, abead can comprise a barcode. A bead can change size, for example, due toswelling in an organic or inorganic solution. A bead can be hydrophobic.A bead can be hydrophilic. A bead can be biocompatible.

A solid support (e.g., a bead) can be visualized. The solid support cancomprise a visualizing tag (e.g., fluorescent dye). A solid support(e.g., a bead) can be etched with an identifier (e.g., a number). Theidentifier can be visualized through imaging the beads.

A solid support can comprise an insoluble, semi-soluble, or insolublematerial. A solid support can be referred to as “functionalized” when itincludes a linker, a scaffold, a building block, or other reactivemoiety attached thereto, whereas a solid support may be“nonfunctionalized” when it lack such a reactive moiety attachedthereto. The solid support can be employed free in solution, such as ina microtiter well format; in a flow-through format, such as in a column;or in a dipstick.

The solid support can comprise a membrane, paper, plastic, coatedsurface, flat surface, glass, slide, chip, or any combination thereof. Asolid support can take the form of resins, gels, microspheres, or othergeometric configurations. A solid support can comprise silica chips,microparticles, nanoparticles, plates, arrays, capillaries, flatsupports such as glass fiber filters, glass surfaces, metal surfaces(steel, gold silver, aluminum, silicon and copper), glass supports,plastic supports, silicon supports, chips, filters, membranes, microwellplates, slides, plastic materials including multiwell plates ormembranes (e.g., formed of polyethylene, polypropylene, polyamide,polyvinylidenedifluoride), and/or wafers, combs, pins or needles (e.g.,arrays of pins suitable for combinatorial synthesis or analysis) orbeads in an array of pits or nanoliter wells of flat surfaces such aswafers (e.g., silicon wafers), wafers with pits with or without filterbottoms.

The solid support can comprise a polymer matrix (e.g., gel, hydrogel).The polymer matrix may be able to permeate intracellular space (e.g.,around organelles). The polymer matrix may able to be pumped throughoutthe circulatory system.

Substrates and Microwell Array

As used herein, a substrate can refer to a type of solid support. Asubstrate can refer to a solid support that can comprise barcodes orstochastic barcodes of the disclosure. A substrate can, for example,comprise a plurality of microwells. For example, a substrate can be awell array comprising two or more microwells. In some embodiments, amicrowell can comprise a small reaction chamber of defined volume. Insome embodiments, a microwell can entrap one or more cells. In someembodiments, a microwell can entrap only one cell. In some embodiments,a microwell can entrap one or more solid supports. In some embodiments,a microwell can entrap only one solid support. In some embodiments, amicrowell entraps a single cell and a single solid support (e.g., abead). A microwell can comprise barcode reagents of the disclosure.

Methods of Barcoding

The disclosure provides for methods for estimating the number ofdistinct targets at distinct locations in a physical sample (e.g.,tissue, organ, tumor, cell). The methods can comprise placing barcodes(e.g., stochastic barcodes) in close proximity with the sample, lysingthe sample, associating distinct targets with the barcodes, amplifyingthe targets and/or digitally counting the targets. The method canfurther comprise analyzing and/or visualizing the information obtainedfrom the spatial labels on the barcodes. In some embodiments, a methodcomprises visualizing the plurality of targets in the sample. Mappingthe plurality of targets onto the map of the sample can includegenerating a two dimensional map or a three dimensional map of thesample. The two dimensional map and the three dimensional map can begenerated prior to or after barcoding (e.g., stochastically barcoding)the plurality of targets in the sample. Visualizing the plurality oftargets in the sample can include mapping the plurality of targets ontoa map of the sample. Mapping the plurality of targets onto the map ofthe sample can include generating a two dimensional map or a threedimensional map of the sample. The two dimensional map and the threedimensional map can be generated prior to or after barcoding theplurality of targets in the sample. in some embodiments, the twodimensional map and the three dimensional map can be generated before orafter lysing the sample. Lysing the sample before or after generatingthe two dimensional map or the three dimensional map can include heatingthe sample, contacting the sample with a detergent, changing the pH ofthe sample, or any combination thereof.

In some embodiments, barcoding the plurality of targets compriseshybridizing a plurality of barcodes with a plurality of targets tocreate barcoded targets (e.g., stochastically barcoded targets).Barcoding the plurality of targets can comprise generating an indexedlibrary of the barcoded targets. Generating an indexed library of thebarcoded targets can be performed with a solid support comprising theplurality of barcodes (e.g., stochastic barcodes).

Contacting a Sample and a Barcode

The disclosure provides for methods for contacting a sample (e.g.,cells) to a substrate of the disclosure. A sample comprising, forexample, a cell, organ, or tissue thin section, can be contacted tobarcodes (e.g., stochastic barcodes). The cells can be contacted, forexample, by gravity flow wherein the cells can settle and create amonolayer. The sample can be a tissue thin section. The thin section canbe placed on the substrate. The sample can be one-dimensional (e.g.,forms a planar surface). The sample (e.g., cells) can be spread acrossthe substrate, for example, by growing/culturing the cells on thesubstrate.

When barcodes are in close proximity to targets, the targets canhybridize to the barcode. The barcodes can be contacted at anon-depletable ratio such that each distinct target can associate with adistinct barcode of the disclosure. To ensure efficient associationbetween the target and the barcode, the targets can be cross-linked tobarcode.

Cell Lysis

Following the distribution of cells and barcodes, the cells can be lysedto liberate the target molecules. Cell lysis can be accomplished by anyof a variety of means, for example, by chemical or biochemical means, byosmotic shock, or by means of thermal lysis, mechanical lysis, oroptical lysis. Cells can be lysed by addition of a cell lysis buffercomprising a detergent (e.g., SDS, Li dodecyl sulfate, Triton X-100,Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), ordigestive enzymes (e.g., proteinase K, pepsin, or trypsin), or anycombination thereof. To increase the association of a target and abarcode, the rate of the diffusion of the target molecules can bealtered by for example, reducing the temperature and/or increasing theviscosity of the lysate.

In some embodiments, the sample can be lysed using a filter paper. Thefilter paper can be soaked with a lysis buffer on top of the filterpaper. The filter paper can be applied to the sample with pressure whichcan facilitate lysis of the sample and hybridization of the targets ofthe sample to the substrate.

In some embodiments, lysis can be performed by mechanical lysis, heatlysis, optical lysis, and/or chemical lysis. Chemical lysis can includethe use of digestive enzymes such as proteinase K, pepsin, and trypsin.Lysis can be performed by the addition of a lysis buffer to thesubstrate. A lysis buffer can comprise Tris HCl. A lysis buffer cancomprise at least about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris HCl. Alysis buffer can comprise at most about 0.01, 0.05, 0.1, 0.5, or 1 M ormore Tris HCL. A lysis buffer can comprise about 0.1 M Tris HCl. The pHof the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more. The pH of the lysis buffer can be at most about 1, 2, 3, 4, 5,6, 7, 8, 9,10, or more. In some embodiments, the pH of the lysis bufferis about 7.5. The lysis buffer can comprise a salt (e.g., LiCl). Theconcentration of salt in the lysis buffer can be at least about 0.1,0.5, or 1 M or more. The concentration of salt in the lysis buffer canbe at most about 0.1, 0.5, or 1 M or more. In some embodiments, theconcentration of salt in the lysis buffer is about 0.5M. The lysisbuffer can comprise a detergent (e.g., SDS, Li dodecyl sulfate, tritonX, tween, NP-40). The concentration of the detergent in the lysis buffercan be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%,0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or more. The concentration ofthe detergent in the lysis buffer can be at most about 0.0001%, 0.0005%,0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%,or more. In some embodiments, the concentration of the detergent in thelysis buffer is about 1% Li dodecyl sulfate. The time used in the methodfor lysis can be dependent on the amount of detergent used. In someembodiments, the more detergent used, the less time needed for lysis.The lysis buffer can comprise a chelating agent (e.g., EDTA, EGTA). Theconcentration of a chelating agent in the lysis buffer can be at leastabout 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration of achelating agent in the lysis buffer can be at most about 1, 5, 10, 15,20, 25, or 30 mM or more. In some embodiments, the concentration ofchelating agent in the lysis buffer is about 10 mM. The lysis buffer cancomprise a reducing reagent (e.g., beta-mercaptoethanol, DTT). Theconcentration of the reducing reagent in the lysis buffer can be atleast about 1, 5, 10, 15, or 20 mM or more. The concentration of thereducing reagent in the lysis buffer can be at most about 1, 5, 10, 15,or 20 mM or more. In some embodiments, the concentration of reducingreagent in the lysis buffer is about 5 mM. In some embodiments, a lysisbuffer can comprise about 0.1M TrisHCl, about pH 7.5, about 0.5M LiCl,about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

Lysis can be performed at a temperature of about 4, 10, 15, 20, 25, or30° C. Lysis can be performed for about 1, 5, 10, 15, or 20 or moreminutes. A lysed cell can comprise at least about 100000, 200000,300000, 400000, 500000, 600000, or 700000 or more target nucleic acidmolecules. A lysed cell can comprise at most about 100000, 200000,300000, 400000, 500000, 600000, or 700000 or more target nucleic acidmolecules.

Attachment of Barcodes to Target Nucleic Acid Molecules

Following lysis of the cells and release of nucleic acid moleculestherefrom, the nucleic acid molecules can randomly associate with thebarcodes of the co-localized solid support. Association can comprisehybridization of a barcode's target recognition region to acomplementary portion of the target nucleic acid molecule (e.g.,oligo(dT) of the barcode can interact with a poly(A) tail of a target).The assay conditions used for hybridization (e.g., buffer pH, ionicstrength, temperature, etc.) can be chosen to promote formation ofspecific, stable hybrids. In some embodiments, the nucleic acidmolecules released from the lysed cells can associate with the pluralityof probes on the substrate (e.g., hybridize with the probes on thesubstrate). When the probes comprise oligo(dT), mRNA molecules canhybridize to the probes and be reverse transcribed. The oligo(dT)portion of the oligonucleotide can act as a primer for first strandsynthesis of the cDNA molecule. For example, in a non-limiting exampleof barcoding illustrated in FIG. 2, at block 216, mRNA molecules canhybridize to barcodes on beads. For example, single-stranded nucleotidefragments can hybridize to the target-binding regions of barcodes.

Attachment can further comprise ligation of a barcode's targetrecognition region and a portion of the target nucleic acid molecule.For example, the target binding region can comprise a nucleic acidsequence that can be capable of specific hybridization to a restrictionsite overhang (e.g., nn EcoRI sticky-end overhang). The assay procedurecan further comprise treating the target nucleic acids with arestriction enzyme (e.g., EcoRI) to create a restriction site overhang.The barcode can then be ligated to any nucleic acid molecule comprisinga sequence complementary to the restriction site overhang. A ligase(e.g., T4 DNA ligase) can be used to join the two fragments.

For example, in a non-limiting example of barcoding illustrated in FIG.2, at block 220, the labeled targets from a plurality of cells (or aplurality of samples) (e.g., target-barcode molecules) can besubsequently pooled, for example, into a tube. The labeled targets canbe pooled by, for example, retrieving the barcodes and/or the beads towhich the target-barcode molecules are attached.

The retrieval of solid support-based collections of attachedtarget-barcode molecules can be implemented by use of magnetic beads andan externally-applied magnetic field. Once the target-barcode moleculeshave been pooled, all further processing can proceed in a singlereaction vessel. Further processing can include, for example, reversetranscription reactions, amplification reactions, cleavage reactions,dissociation reactions, and/or nucleic acid extension reactions. Furtherprocessing reactions can be performed within the microwells, that is,without first pooling the labeled target nucleic acid molecules from aplurality of cells.

Reverse Transcription

The disclosure provides for a method to create a target-barcodeconjugate using reverse transcription (e.g., at block 224 of FIG. 2).The target-barcode conjugate can comprise the barcode and acomplementary sequence of all or a portion of the target nucleic acid(i.e., a barcoded cDNA molecule, such as a stochastically barcoded cDNAmolecule). Reverse transcription of the associated RNA molecule canoccur by the addition of a reverse transcription primer along with thereverse transcriptase. The reverse transcription primer can be anoligo(dT) primer, a random hexanucleotide primer, or a target-specificoligonucleotide primer. Oligo(dT) primers can be, or can be about, 12-18nucleotides in length and bind to the endogenous poly(A) tail at the 3′end of mammalian mRNA. Random hexanucleotide primers can bind to mRNA ata variety of complementary sites. Target-specific oligonucleotideprimers typically selectively prime the mRNA of interest.

In some embodiments, reverse transcription of the labeled-RNA moleculecan occur by the addition of a reverse transcription primer. In someembodiments, the reverse transcription primer is an oligo(dT) primer,random hexanucleotide primer, or a target-specific oligonucleotideprimer. Generally, oligo(dT) primers are 12-18 nucleotides in length andbind to the endogenous poly(A) tail at the 3′ end of mammalian mRNA.Random hexanucleotide primers can bind to mRNA at a variety ofcomplementary sites. Target-specific oligonucleotide primers typicallyselectively prime the mRNA of interest.

Reverse transcription can occur repeatedly to produce multiplelabeled-cDNA molecules. The methods disclosed herein can compriseconducting at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 reverse transcription reactions. The methodcan comprise conducting at least about 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

Amplification

One or more nucleic acid amplification reactions (e.g., at block 228 ofFIG. 2) can be performed to create multiple copies of the labeled targetnucleic acid molecules. Amplification can be performed in a multiplexedmanner, wherein multiple target nucleic acid sequences are amplifiedsimultaneously. The amplification reaction can be used to add sequencingadaptors to the nucleic acid molecules. The amplification reactions cancomprise amplifying at least a portion of a sample label, if present.The amplification reactions can comprise amplifying at least a portionof the cellular label and/or barcode sequence (e.g., a molecular label).The amplification reactions can comprise amplifying at least a portionof a sample tag, a cell label, a spatial label, a barcode sequence(e.g., a molecular label), a target nucleic acid, or a combinationthereof. The amplification reactions can comprise amplifying 0.5%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, or a rangeor a number between any two of these values, of the plurality of nucleicacids. The method can further comprise conducting one or more cDNAsynthesis reactions to produce one or more cDNA copies of target-barcodemolecules comprising a sample label, a cell label, a spatial label,and/or a barcode sequence (e.g., a a molecular label).

In some embodiments, amplification can be performed using a polymerasechain reaction (PCR). As used herein, PCR can refer to a reaction forthe in vitro amplification of specific DNA sequences by the simultaneousprimer extension of complementary strands of DNA. As used herein, PCRcan encompass derivative forms of the reaction, including but notlimited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR,multiplexed PCR, digital PCR, and assembly PCR.

Amplification of the labeled nucleic acids can comprise non-PCR basedmethods. Examples of non-PCR based methods include, but are not limitedto, multiple displacement amplification (MDA), transcription-mediatedamplification (TMA), nucleic acid sequence-based amplification (NASBA),strand displacement amplification (SDA), real-time SDA, rolling circleamplification, or circle-to-circle amplification. Other non-PCR-basedamplification methods include multiple cycles of DNA-dependent RNApolymerase-driven RNA transcription amplification or RNA-directed DNAsynthesis and transcription to amplify DNA or RNA targets, a ligasechain reaction (LCR), and a Qβ replicase (Qβ) method, use of palindromicprobes, strand displacement amplification, oligonucleotide-drivenamplification using a restriction endonuclease, an amplification methodin which a primer is hybridized to a nucleic acid sequence and theresulting duplex is cleaved prior to the extension reaction andamplification, strand displacement amplification using a nucleic acidpolymerase lacking 5′ exonuclease activity, rolling circleamplification, and ramification extension amplification (RAM). In someembodiments, the amplification does not produce circularizedtranscripts.

In some embodiments, the methods disclosed herein further compriseconducting a polymerase chain reaction on the labeled nucleic acid(e.g., labeled-RNA, labeled-DNA, labeled-cDNA) to produce a labeledamplicon (e.g., a stochastically labeledamplicon). The labeled ampliconcan be double-stranded molecule. The double-stranded molecule cancomprise a double-stranded RNA molecule, a double-stranded DNA molecule,or a RNA molecule hybridized to a DNA molecule. One or both of thestrands of the double-stranded molecule can comprise a sample label, aspatial label, a cell label, and/or a barcode sequence (e.g., amolecular label). The labeled amplicon can be a single-strandedmolecule. The single-stranded molecule can comprise DNA, RNA, or acombination thereof. The nucleic acids of the disclosure can comprisesynthetic or altered nucleic acids.

Amplification can comprise use of one or more non-natural nucleotides.Non-natural nucleotides can comprise photolabile or triggerablenucleotides. Examples of non-natural nucleotides can include, but arenot limited to, peptide nucleic acid (PNA), morpholino and lockednucleic acid (LNA), as well as glycol nucleic acid (GNA) and threosenucleic acid (TNA). Non-natural nucleotides can be added to one or morecycles of an amplification reaction. The addition of the non-naturalnucleotides can be used to identify products as specific cycles or timepoints in the amplification reaction.

Conducting the one or more amplification reactions can comprise the useof one or more primers. The one or more primers can comprise, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or morenucleotides. The one or more primers can comprise at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one ormore primers can comprise less than 12-15 nucleotides. The one or moreprimers can anneal to at least a portion of the plurality of labeledtargets (e.g., stochastically labeled targets). The one or more primerscan anneal to the 3′ end or 5′ end of the plurality of labeled targets.The one or more primers can anneal to an internal region of theplurality of labeled targets. The internal region can be at least about50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3′ endsthe plurality of labeled targets. The one or more primers can comprise afixed panel of primers. The one or more primers can comprise at leastone or more custom primers. The one or more primers can comprise atleast one or more control primers. The one or more primers can compriseat least one or more gene-specific primers.

The one or more primers can comprise a universal primer. The universalprimer can anneal to a universal primer binding site. The one or morecustom primers can anneal to a first sample label, a second samplelabel, a spatial label, a cell label, a barcode sequence (e.g., amolecular label), a target, or any combination thereof. The one or moreprimers can comprise a universal primer and a custom primer. The customprimer can be designed to amplify one or more targets. The targets cancomprise a subset of the total nucleic acids in one or more samples. Thetargets can comprise a subset of the total labeled targets in one ormore samples. The one or more primers can comprise at least 96 or morecustom primers. The one or more primers can comprise at least 960 ormore custom primers. The one or more primers can comprise at least 9600or more custom primers. The one or more custom primers can anneal to twoor more different labeled nucleic acids. The two or more differentlabeled nucleic acids can correspond to one or more genes.

Any amplification scheme can be used in the methods of the presentdisclosure. For example, in one scheme, the first round PCR can amplifymolecules attached to the bead using a gene specific primer and a primeragainst the universal Illumina sequencing primer 1 sequence. The secondround of PCR can amplify the first PCR products using a nested genespecific primer flanked by Illumina sequencing primer 2 sequence, and aprimer against the universal Illumina sequencing primer 1 sequence. Thethird round of PCR adds P5 and P7 and sample index to turn PCR productsinto an Illumina sequencing library. Sequencing using 150 bp×2sequencing can reveal the cell label and barcode sequence (e.g.,molecular label) on read 1, the gene on read 2, and the sample index onindex 1 read.

In some embodiments, nucleic acids can be removed from the substrateusing chemical cleavage. For example, a chemical group or a modifiedbase present in a nucleic acid can be used to facilitate its removalfrom a solid support. For example, an enzyme can be used to remove anucleic acid from a substrate. For example, a nucleic acid can beremoved from a substrate through a restriction endonuclease digestion.For example, treatment of a nucleic acid containing a dUTP or ddUTP withuracil-d-glycosylase (UDG) can be used to remove a nucleic acid from asubstrate. For example, a nucleic acid can be removed from a substrateusing an enzyme that performs nucleotide excision, such as a baseexcision repair enzyme, such as an apurinic/apyrimidinic (AP)endonuclease. In some embodiments, a nucleic acid can be removed from asubstrate using a photocleavable group and light. In some embodiments, acleavable linker can be used to remove a nucleic acid from thesubstrate. For example, the cleavable linker can comprise at least oneof biotin/avidin, biotin/streptavidin, biotin/neutravidin, Ig-protein A,a photo-labile linker, acid or base labile linker group, or an aptamer.

When the probes are gene-specific, the molecules can hybridize to theprobes and be reverse transcribed and/or amplified. In some embodiments,after the nucleic acid has been synthesized (e.g., reverse transcribed),it can be amplified. Amplification can be performed in a multiplexmanner, wherein multiple target nucleic acid sequences are amplifiedsimultaneously. Amplification can add sequencing adaptors to the nucleicacid.

In some embodiments, amplification can be performed on the substrate,for example, with bridge amplification. cDNAs can be homopolymer tailedin order to generate a compatible end for bridge amplification usingoligo(dT) probes on the substrate. In bridge amplification, the primerthat is complementary to the 3′ end of the template nucleic acid can bethe first primer of each pair that is covalently attached to the solidparticle. When a sample containing the template nucleic acid iscontacted with the particle and a single thermal cycle is performed, thetemplate molecule can be annealed to the first primer and the firstprimer is elongated in the forward direction by addition of nucleotidesto form a duplex molecule consisting of the template molecule and anewly formed DNA strand that is complementary to the template. In theheating step of the next cycle, the duplex molecule can be denatured,releasing the template molecule from the particle and leaving thecomplementary DNA strand attached to the particle through the firstprimer. In the annealing stage of the annealing and elongation step thatfollows, the complementary strand can hybridize to the second primer,which is complementary to a segment of the complementary strand at alocation removed from the first primer. This hybridization can cause thecomplementary strand to form a bridge between the first and secondprimers secured to the first primer by a covalent bond and to the secondprimer by hybridization. In the elongation stage, the second primer canbe elongated in the reverse direction by the addition of nucleotides inthe same reaction mixture, thereby converting the bridge to adouble-stranded bridge. The next cycle then begins, and thedouble-stranded bridge can be denatured to yield two single-strandednucleic acid molecules, each having one end attached to the particlesurface via the first and second primers, respectively, with the otherend of each unattached. In the annealing and elongation step of thissecond cycle, each strand can hybridize to a further complementaryprimer, previously unused, on the same particle, to form newsingle-strand bridges. The two previously unused primers that are nowhybridized elongate to convert the two new bridges to double-strandbridges.

The amplification reactions can comprise amplifying at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of theplurality of nucleic acids.

Amplification of the labeled nucleic acids can comprise PCR-basedmethods or non-PCR based methods. Amplification of the labeled nucleicacids can comprise exponential amplification of the labeled nucleicacids. Amplification of the labeled nucleic acids can comprise linearamplification of the labeled nucleic acids. Amplification can beperformed by polymerase chain reaction (PCR). PCR can refer to areaction for the in vitro amplification of specific DNA sequences by thesimultaneous primer extension of complementary strands of DNA. PCR canencompass derivative forms of the reaction, including but not limitedto, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexedPCR, digital PCR, suppression PCR, semi-suppressive PCR and assemblyPCR.

In some embodiments, amplification of the labeled nucleic acidscomprises non-PCR based methods. Examples of non-PCR based methodsinclude, but are not limited to, multiple displacement amplification(MDA), transcription-mediated amplification (TMA), nucleic acidsequence-based amplification (NASBA), strand displacement amplification(SDA), real-time SDA, rolling circle amplification, or circle-to-circleamplification. Other non-PCR-based amplification methods includemultiple cycles of DNA-dependent RNA polymerase-driven RNA transcriptionamplification or RNA-directed DNA synthesis and transcription to amplifyDNA or RNA targets, a ligase chain reaction (LCR), a Qβ replicase (Qβ),use of palindromic probes, strand displacement amplification,oligonucleotide-driven amplification using a restriction endonuclease,an amplification method in which a primer is hybridized to a nucleicacid sequence and the resulting duplex is cleaved prior to the extensionreaction and amplification, strand displacement amplification using anucleic acid polymerase lacking 5′ exonuclease activity, rolling circleamplification, and/or ramification extension amplification (RAM).

In some embodiments, the methods disclosed herein further compriseconducting a nested polymerase chain reaction on the amplified amplicon(e.g., target). The amplicon can be double-stranded molecule. Thedouble-stranded molecule can comprise a double-stranded RNA molecule, adouble-stranded DNA molecule, or a RNA molecule hybridized to a DNAmolecule. One or both of the strands of the double-stranded molecule cancomprise a sample tag or molecular identifier label. Alternatively, theamplicon can be a single-stranded molecule. The single-stranded moleculecan comprise DNA, RNA, or a combination thereof. The nucleic acids ofthe present invention can comprise synthetic or altered nucleic acids.

In some embodiments, the method comprises repeatedly amplifying thelabeled nucleic acid to produce multiple amplicons. The methodsdisclosed herein can comprise conducting at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplificationreactions. Alternatively, the method comprises conducting at least about25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100amplification reactions.

Amplification can further comprise adding one or more control nucleicacids to one or more samples comprising a plurality of nucleic acids.Amplification can further comprise adding one or more control nucleicacids to a plurality of nucleic acids. The control nucleic acids cancomprise a control label.

Amplification can comprise use of one or more non-natural nucleotides.Non-natural nucleotides can comprise photolabile and/or triggerablenucleotides. Examples of non-natural nucleotides include, but are notlimited to, peptide nucleic acid (PNA), morpholino and locked nucleicacid (LNA), as well as glycol nucleic acid (GNA) and threose nucleicacid (TNA). Non-natural nucleotides can be added to one or more cyclesof an amplification reaction. The addition of the non-naturalnucleotides can be used to identify products as specific cycles or timepoints in the amplification reaction.

Conducting the one or more amplification reactions can comprise the useof one or more primers. The one or more primers can comprise one or moreoligonucleotides. The one or more oligonucleotides can comprise at leastabout 7-9 nucleotides. The one or more oligonucleotides can compriseless than 12-15 nucleotides. The one or more primers can anneal to atleast a portion of the plurality of labeled nucleic acids. The one ormore primers can anneal to the 3′ end and/or 5′ end of the plurality oflabeled nucleic acids. The one or more primers can anneal to an internalregion of the plurality of labeled nucleic acids. The internal regioncan be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000nucleotides from the 3′ ends the plurality of labeled nucleic acids. Theone or more primers can comprise a fixed panel of primers. The one ormore primers can comprise at least one or more custom primers. The oneor more primers can comprise at least one or more control primers. Theone or more primers can comprise at least one or more housekeeping geneprimers. The one or more primers can comprise a universal primer. Theuniversal primer can anneal to a universal primer binding site. The oneor more custom primers can anneal to the first sample tag, the secondsample tag, the molecular identifier label, the nucleic acid or aproduct thereof. The one or more primers can comprise a universal primerand a custom primer. The custom primer can be designed to amplify one ormore target nucleic acids. The target nucleic acids can comprise asubset of the total nucleic acids in one or more samples. In someembodiments, the primers are the probes attached to the array of thedisclosure.

In some embodiments, barcoding (e.g., stochastically barcoding) theplurality of targets in the sample further comprises generating anindexed library of the barcoded targets (e.g., stochastically barcodedtargets) or barcoded fragments of the targets. The barcode sequences ofdifferent barcodes (e.g., the molecular labels of different stochasticbarcodes) can be different from one another. Generating an indexedlibrary of the barcoded targets includes generating a plurality ofindexed polynucleotides from the plurality of targets in the sample. Forexample, for an indexed library of the barcoded targets comprising afirst indexed target and a second indexed target, the label region ofthe first indexed polynucleotide can differ from the label region of thesecond indexed polynucleotide by, by about, by at least, or by at most,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or a number or a rangebetween any two of these values, nucleotides. In some embodiments,generating an indexed library of the barcoded targets includescontacting a plurality of targets, for example mRNA molecules, with aplurality of oligonucleotides including a poly(T) region and a labelregion; and conducting a first strand synthesis using a reversetranscriptase to produce single-strand labeled cDNA molecules eachcomprising a cDNA region and a label region, wherein the plurality oftargets includes at least two mRNA molecules of different sequences andthe plurality of oligonucleotides includes at least two oligonucleotidesof different sequences. Generating an indexed library of the barcodedtargets can further comprise amplifying the single-strand labeled cDNAmolecules to produce double-strand labeled cDNA molecules; andconducting nested PCR on the double-strand labeled cDNA molecules toproduce labeled amplicons. In some embodiments, the method can includegenerating an adaptor-labeled amplicon.

Barcoding (e.g., stochastic barcoding) can include using nucleic acidbarcodes or tags to label individual nucleic acid (e.g., DNA or RNA)molecules. In some embodiments, it involves adding DNA barcodes or tagsto cDNA molecules as they are generated from mRNA. Nested PCR can beperformed to minimize PCR amplification bias. Adaptors can be added forsequencing using, for example, next generation sequencing (NGS). Thesequencing results can be used to determine cell labels, molecularlabels, and sequences of nucleotide fragments of the one or more copiesof the targets, for example at block 232 of FIG. 2.

FIG. 3 is a schematic illustration showing a non-limiting exemplaryprocess of generating an indexed library of the barcoded targets (e.g.,stochastically barcoded targets), such as barcoded mRNAs or fragmentsthereof. As shown in step 1, the reverse transcription process canencode each mRNA molecule with a unique molecular label, a cell label,and a universal PCR site. In particular, RNA molecules 302 can bereverse transcribed to produce labeled cDNA molecules 304, including acDNA region 306, by hybridization (e.g., stochastic hybridization) of aset of barcodes (e.g., stochastic barcodes) 310 to the poly(A) tailregion 308 of the RNA molecules 302. Each of the barcodes 310 cancomprise a target-binding region, for example a poly(dT) region 312, alabel region 314 (e.g., a barcode sequence or a molecule), and auniversal PCR region 316.

In some embodiments, the cell label can include 3 to 20 nucleotides. Insome embodiments, the molecular label can include 3 to 20 nucleotides.In some embodiments, each of the plurality of stochastic barcodesfurther comprises one or more of a universal label and a cell label,wherein universal labels are the same for the plurality of stochasticbarcodes on the solid support and cell labels are the same for theplurality of stochastic barcodes on the solid support. In someembodiments, the universal label can include 3 to 20 nucleotides. Insome embodiments, the cell label comprises 3 to 20 nucleotides.

In some embodiments, the label region 314 can include a barcode sequenceor a molecular label 318 and a cell label 320. In some embodiments, thelabel region 314 can include one or more of a universal label, adimension label, and a cell label. The barcode sequence or molecularlabel 318 can be, can be about, can be at least, or can be at most, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or anumber or a range between any of these values, of nucleotides in length.The cell label 320 can be, can be about, can be at least, or can be atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or a number or a range between any of these values, of nucleotidesin length. The universal label can be, can be about, can be at least, orcan be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, or a number or a range between any of these values, ofnucleotides in length. Universal labels can be the same for theplurality of stochastic barcodes on the solid support and cell labelsare the same for the plurality of stochastic barcodes on the solidsupport. The dimension label can be, can be about, can be at least, orcan be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, or a number or a range between any of these values, ofnucleotides in length.

In some embodiments, the label region 314 can comprise, comprise about,comprise at least, or comprise at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, or a number or a range between any of these values, differentlabels, such as a barcode sequence or a molecular label 318 and a celllabel 320. Each label can be, can be about, can be at least, or can beat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or a number or a range between any of these values, of nucleotidesin length. A set of barcodes or stochastic barcodes 310 can contain,contain about, contain at least, or can be at most, 10, 20, 40, 50, 70,80, 90, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10²⁰, or a number or a range between any of these values,barcodes or stochastic barcodes 310. And the set of barcodes orstochastic barcodes 310 can, for example, each contain a unique labelregion 314. The labeled cDNA molecules 304 can be purified to removeexcess barcodes or stochastic barcodes 310. Purification can compriseAmpure bead purification.

As shown in step 2, products from the reverse transcription process instep 1 can be pooled into 1 tube and PCR amplified with a 1^(st) PCRprimer pool and a 1^(st) universal PCR primer. Pooling is possiblebecause of the unique label region 314. In particular, the labeled cDNAmolecules 304 can be amplified to produce nested PCR labeled amplicons322. Amplification can comprise multiplex PCR amplification.Amplification can comprise a multiplex PCR amplification with 96multiplex primers in a single reaction volume. In some embodiments,multiplex PCR amplification can utilize, utilize about, utilize atleast, or utilize at most, 10, 20, 40, 50, 70, 80, 90, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10²⁰, or anumber or a range between any of these values, multiplex primers in asingle reaction volume. Amplification can comprise using a 1^(st) PCRprimer pool 324 comprising custom primers 326A-C targeting specificgenes and a universal primer 328. The custom primers 326 can hybridizeto a region within the cDNA portion 306′ of the labeled cDNA molecule304. The universal primer 328 can hybridize to the universal PCR region316 of the labeled cDNA molecule 304.

As shown in step 3 of FIG. 3, products from PCR amplification in step 2can be amplified with a nested PCR primers pool and a 2^(nd) universalPCR primer. Nested PCR can minimize PCR amplification bias. Inparticular, the nested PCR labeled amplicons 322 can be furtheramplified by nested PCR. The nested PCR can comprise multiplex PCR withnested PCR primers pool 330 of nested PCR primers 332 a-c and a 2^(nd)universal PCR primer 328′ in a single reaction volume. The nested PCRprimer pool 328 can contain, contain about, contain at least, or containat most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or arange between any of these values, different nested PCR primers 330. Thenested PCR primers 332 can contain an adaptor 334 and hybridize to aregion within the cDNA portion 306″ of the labeled amplicon 322. Theuniversal primer 328′ can contain an adaptor 336 and hybridize to theuniversal PCR region 316 of the labeled amplicon 322. Thus, step 3produces adaptor-labeled amplicon 338. In some embodiments, nested PCRprimers 332 and the 2^(nd) universal PCR primer 328′ may not contain theadaptors 334 and 336. The adaptors 334 and 336 can instead be ligated tothe products of nested PCR to produce adaptor-labeled amplicon 338.

As shown in step 4, PCR products from step 3 can be PCR amplified forsequencing using library amplification primers. In particular, theadaptors 334 and 336 can be used to conduct one or more additionalassays on the adaptor-labeled amplicon 338. The adaptors 334 and 336 canbe hybridized to primers 340 and 342. The one or more primers 340 and342 can be PCR amplification primers. The one or more primers 340 and342 can be sequencing primers. The one or more adaptors 334 and 336 canbe used for further amplification of the adaptor-labeled amplicons 338.The one or more adaptors 334 and 336 can be used for sequencing theadaptor-labeled amplicon 338. The primer 342 can contain a plate index344 so that amplicons generated using the same set of barcodes orstochastic barcodes 310 can be sequenced in one sequencing reactionusing next generation sequencing (NGS).

Compositions Comprising Cellular Component Binding Reagents Associatedwith Oligonucleotides

Some embodiments disclosed herein provide a plurality of compositionseach comprising a cellular component binding reagent (such as a proteinbinding reagent) that is conjugated with an oligonucleotide, wherein theoligonucleotide comprises a unique identifier for the cellular componentbinding reagent that it is conjugated with. In some embodiments, thecellular component binding reagent is capable of specifically binding toa cellular component target. For example, a binding target of thecellular component binding reagent can be, or comprise, a carbohydrate,a lipid, a protein, an extracellular protein, a cell-surface protein, acell marker, a B-cell receptor, a T-cell receptor, a majorhistocompatibility complex, a tumor antigen, a receptor, an integrin, anintracellular protein, or any combination thereof. In some embodiments,the cellular component binding reagent (e.g., a protein binding reagent)is capable of specifically binding to an antigen target or a proteintarget. In some embodiments, each of the oligonucleotides can comprise abarcode, such as a stochastic barcode. A barcode can comprise a barcodesequence (e.g., a molecular label), a cell label, a sample label, or anycombination thereof. In some embodiments, each of the oligonucleotidescan comprise a linker. In some embodiments, each of the oligonucleotidescan comprise a binding site for an oligonucleotide probe, such as apoly(A) tail. For example, the poly(A) tail can be, e.g., unanchored toa solid support or anchored to a solid support. The poly(A) tail can befrom about 10 to 50 nucleotides in length. In some embodiments, thepoly(A) tail can be 18 nucleotides in length. The oligonucleotides cancomprise deoxyribonucleotides, ribonucleotides, or both.

The unique identifiers can be, for example, a nucleotide sequence havingany suitable length, for example, from about 4 nucleotides to about 200nucleotides. In some embodiments, the unique identifier is a nucleotidesequence of 25 nucleotides to about 45 nucleotides in length. In someembodiments, the unique identifier can have a length that is, is about,is less than, is greater than, 4 nucleotides, 5 nucleotides, 6nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50nucleotides, 55 nucleotides, 60 nucleotides, 70 nucleotides, 80nucleotides, 90 nucleotides, 100 nucleotides, 200 nucleotides, or arange that is between any two of the above values.

In some embodiments, the unique identifiers are selected from a diverseset of unique identifiers. The diverse set of unique identifiers cancomprise, or comprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or arange between any two of these values, different unique identifiers. Thediverse set of unique identifiers can comprise at least, or comprise atmost, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, or 5000, different unique identifiers. In someembodiments, the set of unique identifiers is designed to have minimalsequence homology to the DNA or RNA sequences of the sample to beanalyzed. In some embodiments, the sequences of the set of uniqueidentifiers are different from each other, or the complement thereof,by, or by about, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides,9 nucleotides, 10 nucleotides, or a number or a range between any two ofthese values. In some embodiments, the sequences of the set of uniqueidentifiers are different from each other, or the complement thereof, byat least, or by at most, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides,9 nucleotides, 10 nucleotides. In some embodiments, the sequences of theset of unique identifiers are different from each other, or thecomplement thereof, by at least 3%, at least 5%, at least 8%, at least10%, at least 15%, at least 20%, or more.

In some embodiments, the unique identifiers can comprise a binding sitefor a primer, such as universal primer. In some embodiments, the uniqueidentifiers can comprise at least two binding sites for a primer, suchas a universal primer. In some embodiments, the unique identifiers cancomprise at least three binding sites for a primer, such as a universalprimer. The primers can be used for amplification of the uniqueidentifiers, for example, by PCR amplification. In some embodiments, theprimers can be used for nested PCR reactions.

Any suitable cellular component binding reagents are contemplated inthis disclosure, such as protein binding reagents, antibodies orfragments thereof, aptamers, small molecules, ligands, peptides,oligonucleotides, etc., or any combination thereof. In some embodiments,the cellular component binding reagents can be polyclonal antibodies,monoclonal antibodies, recombinant antibodies, single chain antibody(sc-Ab), or fragments thereof, such as Fab, Fv, etc. In someembodiments, the plurality of cellular component binding reagents cancomprise, or comprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or arange between any two of these values, different cellular componentreagents. In some embodiments, the plurality of cellular componentbinding reagents can comprise at least, or comprise at most, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000, or 5000, different cellular component reagents.

The oligonucleotide can be conjugated with the cellular componentbinding reagent through various mechanism. In some embodiments, theoligonucleotide can be conjugated with the cellular component bindingreagent covalently. In some embodiment, the oligonucleotide can beconjugated with the cellular component binding reagent non-covalently.In some embodiments, the oligonucleotide is conjugated with the cellularcomponent binding reagent through a linker. The linker can be, forexample, cleavable or detachable from the cellular component bindingreagent and/or the oligonucleotide. In some embodiments, the linker cancomprise a chemical group that reversibly attaches the oligonucleotideto the cellular component binding reagents. The chemical group can beconjugated to the linker, for example, through an amine group. In someembodiments, the linker can comprise a chemical group that forms astable bond with another chemical group conjugated to the cellularcomponent binding reagent. For example, the chemical group can be a UVphotocleavable group, a disulfide bond, a streptavidin, a biotin, anamine, etc. In some embodiments, the chemical group can be conjugated tothe cellular component binding reagent through a primary amine on anamino acid, such as lysine, or the N-terminus. Commercially availableconjugation kits, such as the Protein-Oligo Conjugation Kit (Solulink,Inc., San Diego, Calif.), the Thunder-Link® oligo conjugation system(Innova Biosciences, Cambridge, United Kingdom), etc., can be used toconjugate the oligonucleotide to the cellular component binding reagent.

The oligonucleotide can be conjugated to any suitable site of thecellular component binding reagent (e.g., a protein binding reagent), aslong as it does not interfere with the specific binding between thecellular component binding reagent and its cellular component target. Insome embodiments, the cellular component binding reagent is a protein,such as an antibody. In some embodiments, the cellular component bindingreagent is not an antibody. In some embodiments, the oligonucleotide canbe conjugated to the antibody anywhere other than the antigen-bindingsite, for example, the Fc region, the C_(H)1 domain, the C_(H)2 domain,the C_(H)3 domain, the C_(L) domain, etc. Methods of conjugatingoligonucleotides to cellular component binding reagents (e.g.,antibodies) have been previously disclosed, for example, in U.S. Pat.No. 6,531,283, the content of which is hereby expressly incorporated byreference in its entirety. Stoichiometry of oligonucleotide to cellularcomponent binding reagent can be varied. To increase the sensitivity ofdetecting the cellular component binding reagent specificoligonucleotide in sequencing, it may be advantageous to increase theratio of oligonucleotide to cellular component binding reagent duringconjugation. In some embodiments, each cellular component bindingreagent can be conjugated with a single oligonucleotide molecule. Insome embodiments, each cellular component binding reagent can beconjugated with more than one oligonucleotide molecule, for example, atleast, or at most, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, or anumber or a range between any two of these values, oligonucleotidemolecules wherein each of the oligonucleotide molecule comprises thesame, or different, unique identifiers. In some embodiments, eachcellular component binding reagent can be conjugated with more than oneoligonucleotide molecule, for example, at least, or at most, 2, 3, 4, 5,10, 20, 30, 40, 50, 100, 1000, oligonucleotide molecules, wherein eachof the oligonucleotide molecule comprises the same, or different, uniqueidentifiers.

In some embodiments, the plurality of cellular component bindingreagents are capable of specifically binding to a plurality of cellularcomponent targets in a sample, such as a single cell, a plurality ofcells, a tissue sample, a tumor sample, a blood sample, or the like. Insome embodiments, the plurality of cellular component targets comprisesa cell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, an antibody, a major histocompatibility complex, a tumorantigen, a receptor, or any combination thereof. In some embodiments,the plurality of cellular component targets can comprise intracellularcellular components. In some embodiments, the plurality of cellularcomponent targets can comprise intracellular cellular components. Insome embodiments, the plurality of cellular components can be, or beabout, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or a number or a range between any two ofthese values, of all the cellular components (e.g., proteins) in a cellor an organism. In some embodiments, the plurality of cellularcomponents can be at least, or be at most, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%,of all the cellular components (e.g., proteins) in a cell or anorganism. In some embodiments, the plurality of cellular componenttargets can comprise, or comprise about, 2, 3, 4, 5, 10, 20, 30, 40, 50,100, 1000, 10000, or a number or a range between any tow of thesevalues, different cellular component targets. In some embodiments, theplurality of cellular component targets can comprise at least, orcomprise at most, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, 10000,different cellular component targets.

FIG. 4 shows a schematic illustration of an exemplary cellular componentbinding reagent, e.g., an antibody, that is conjugated with anoligonucleotide comprising a unique identifier sequence for theantibody. An oligonucleotide-conjugated with a cellular componentbinding reagent, an oligonucleotide for conjugation with a cellularcomponent binding reagent, or an oligonucleotide previously conjugatedwith a cellular component binding reagent can be referred to herein asan antibody oligonucleotide (abbreviated as a binding reagentoligonucleotide). An oligonucleotide-conjugated with an antibody, anoligonucleotide for conjugation with an antibody, or an oligonucleotidepreviously conjugated with an antibody can be referred to herein as anantibody oligonucleotide (abbreviated as an “AbOligo” or “AbO”). Theoligonucleotide can also comprise additional components, including butnot limited to, one or more linker, one or more unique identifier forthe antibody, optionally one or more barcode sequences (e.g., molecularlabels), and a poly(A) tail. In some embodiments, the oligonucleotidecan comprise, from 5′ to 3′, a linker, a unique identifier, a barcodesequence (e.g., a molecular label), and a poly(A) tail. An antibodyoligonucleotide can be an mRNA mimic.

FIG. 5 shows a schematic illustration of an exemplary cellular componentbinding reagent, e.g., an antibody, that is conjugated with anoligonucleotide comprising a unique identifier sequence for theantibody. The cellular component binding reagent can be capable ofspecifically binding to at least one cellular component target, such asan antigen target or a protein target. A binding reagent oligonucleotide(e.g., a sample indexing oligonucleotide, or an antibodyoligonucleotide) can comprise a sequence (e.g., a sample indexingsequence) for performing the methods of the disclosure. For example, asample indexing oligonucleotide can comprise a sample indexing sequencefor identifying sample origin of one or more cells of a sample. Indexingsequences (e.g., sample indexing sequences) of at least two compositionscomprising two cellular component binding reagents (e.g., sampleindexing compositions) of the plurality of compositions comprisingcellular component binding reagents can comprise different sequences. Insome embodiments, the binding reagent oligonucleotide is not homologousto genomic sequences of a species. The binding reagent oligonucleotidecan be configured to be detachable or non-detachable from the cellularcomponent binding reagent.

The oligonucleotide conjugated to a cellular component binding reagentcan, for example, comprise a barcode sequence (e.g., a molecular labelsequence), a poly(A) tail, or a combination thereof. An oligonucleotideconjugated to a cellular component binding reagent can be an mRNA mimic.In some embodiments, the sample indexing oligonucleotide comprises asequence complementary to a capture sequence of at least one barcode ofthe plurality of barcodes. A target binding region of the barcode cancomprise the capture sequence. The target binding region can, forexample, comprise a poly(dT) region. In some embodiments, the sequenceof the sample indexing oligonucleotide complementary to the capturesequence of the barcode can comprise a poly(A) tail. The sample indexingoligonucleotide can comprise a molecular label.

In some embodiments, the binding reagent oligonucleotide (e.g., thesample oligonucleotide) comprises a nucleotide sequence of, or anucleotide sequence of about, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120,128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530,540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670,680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810,820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,960, 970, 980, 990, 1000, or a number or a range between any two ofthese values, nucleotides in length. In some embodiments, the bindingreagent oligonucleotide comprises a nucleotide sequence of at least, orof at most, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or1000, nucleotides in length.

In some embodiments, the cellular component binding reagent comprises anantibody, a tetramer, an aptamers, a protein scaffold, or a combinationthereof. The binding reagent oligonucleotide can be conjugated to thecellular component binding reagent, for example, through a linker. Thebinding reagent oligonucleotide can comprise the linker. The linker cancomprise a chemical group. The chemical group can be reversibly, orirreversibly, attached to the molecule of the cellular component bindingreagent. The chemical group can be selected from the group consisting ofa UV photocleavable group, a disulfide bond, a streptavidin, a biotin,an amine, and any combination thereof.

In some embodiments, the cellular component binding reagent can bind toADAM10, CD156c, ANO6, ATP1B2, ATP1B3, BSG, CD147, CD109, CD230, CD29,CD298, ATP1B3, CD44, CD45, CD47, CD51, CD59, CD63, CD97, CD98, SLC3A2,CLDND1, HLA-ABC, ICAM1, ITFG3, MPZL1, NA K ATPase alpha1, ATP1A1, NPTN,PMCA ATPase, ATP2B1, SLC1A5, SLC29A1, SLC2A1, SLC44A2, or anycombination thereof.

In some embodiments, the protein target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. In some embodiments, the antigen or protein target is, orcomprises, a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, or any combination thereof. The antigen orprotein target can be, or comprise, a lipid, a carbohydrate, or anycombination thereof. The protein target can be selected from a groupcomprising a number of protein targets. The number of antigen target orprotein targets can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or a numberor a range between any two of these values. The number of proteintargets can be at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000.

The cellular component binding reagent (e.g., a protein binding reagent)can be associated with two or more binding reagent oligonucleotide(e.g., sample indexing oligonucleotides) with an identical sequence. Thecellular component binding reagent can be associated with two or morebinding reagent oligonucleotides with different sequences. The number ofbinding reagent oligonucleotides associated with the cellular componentbinding reagent can be different in different implementations. In someembodiments, the number of binding reagent oligonucleotides, whetherhaving an identical sequence, or different sequences, can be, or beabout, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or arange between any two of these values. In some embodiments, the numberof binding reagent oligonucleotides can be at least, or be at most, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, or 1000.

The plurality of compositions comprising cellular component bindingreagents (e.g., the plurality of sample indexing compositions) cancomprise one or more additional cellular component binding reagents notconjugated with the binding reagent oligonucleotide (such as sampleindexing oligonucleotide), which is also referred to herein as thebinding reagent oligonucleotide-free cellular component binding reagent(such as sample indexing oligonucleotide-free cellular component bindingreagent). The number of additional cellular component binding reagentsin the plurality of compositions can be different in differentimplementations. In some embodiments, the number of additional cellularcomponent binding reagents can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a rangebetween any two of these values. In some embodiments, the number ofadditional cellular component binding reagents can be at least, or be atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or100. The cellular component binding reagent and any of the additionalcellular component binding reagents can be identical, in someembodiments.

In some embodiments, a mixture comprising cellular component bindingreagent(s) that is conjugated with one or more binding reagentoligonucleotides (e.g., sample indexing oligonucleotides) and cellularcomponent binding reagent(s) that is not conjugated with binding reagentoligonucleotides is provided. The mixture can be used in someembodiments of the methods disclosed herein, for example, to contact thesample(s) and/or cell(s). The ratio of (1) the number of a cellularcomponent binding reagent conjugated with a binding reagentoligonucleotide and (2) the number of another cellular component bindingreagent (e.g., the same cellular component binding reagent) notconjugated with the binding reagent oligonucleotide (e.g., sampleindexing oligonucleotide) or other binding reagent oligonucleotide(s) inthe mixture can be different in different implementations. In someembodiments, the ratio can be, or be about, 1:1, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29,1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41,1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53,1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65,1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77,1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89,1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000,1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or anumber or a range between any two of the values. In some embodiments,the ratio can be at least, or be at most, 1:1, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29,1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41,1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53,1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65,1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77,1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89,1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000,1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, or 1:10000.

In some embodiments, the ratio can be, or be about, 1:1, 1.1:1, 1.2:1,1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1,30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1,42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1,54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1,66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1,78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1,90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1,200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1,3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or anumber or a range between any two of the values. In some embodiments,the ratio can be at least, or be at most, 1:1, 1.1:1, 1.2:1, 1.3:1,1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1,30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1,42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1,54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1,66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1,78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1,90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1,200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1,3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, or 10000:1.

A cellular component binding reagent can be conjugated with a bindingreagent oligonucleotide (e.g., a sample indexing oligonucleotide), ornot. In some embodiments, the percentage of the cellular componentbinding reagent conjugated with a binding reagent oligonucleotide (e.g.,a sample indexing oligonucleotide) in a mixture comprising the cellularcomponent binding reagent that is conjugated with the binding reagentoligonucleotide and the cellular component binding reagent(s) that isnot conjugated with the binding reagent oligonucleotide can be, or beabout, 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%,0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two ofthese values. In some embodiments, the percentage of the cellularcomponent binding reagent conjugated with a sample indexingoligonucleotide in a mixture can be at least, or be at most,0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%,0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%.

In some embodiments, the percentage of the cellular component bindingreagent not conjugated with a binding reagent oligonucleotide (e.g., asample indexing oligonucleotide) in a mixture comprising a cellularcomponent binding reagent conjugated with a binding reagentoligonucleotide (e.g., a sample indexing oligonucleotide) and thecellular component binding reagent that is not conjugated with thesample indexing oligonucleotide can be, or be about, 0.000000001%,0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%,0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100%, or a number or a range between any two of these values. In someembodiments, the percentage of the cellular component binding reagentnot conjugated with a binding reagent oligonucleotide in a mixture canbe at least, or be at most, 0.000000001%, 0.00000001%, 0.0000001%,0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

Cellular Component Cocktails

In some embodiments, a cocktail of cellular component binding reagents(e.g., an antibody cocktail) can be used to increase labelingsensitivity in the methods disclosed herein. Without being bound by anyparticular theory, it is believed that this may be because cellularcomponent expression or protein expression can vary between cell typesand cell states, making finding a universal cellular component bindingreagent or antibody that labels all cell types challenging. For example,cocktail of cellular component binding reagents can be used to allow formore sensitive and efficient labeling of more sample types. The cocktailof cellular component binding reagents can include two or more differenttypes of cellular component binding reagents, for example a wider rangeof cellular component binding reagents or antibodies. Cellular componentbinding reagents that label different cellular component targets can bepooled together to create a cocktail that sufficiently labels all celltypes, or one or more cell types of interest.

In some embodiments, each of the plurality of compositions (e.g., sampleindexing compositions) comprises a cellular component binding reagent.In some embodiments, a composition of the plurality of compositionscomprises two or more cellular component binding reagents, wherein eachof the two or more cellular component binding reagents is associatedwith a binding reagent oligonucleotide (e.g., a sample indexingoligonucleotide), wherein at least one of the two or more cellularcomponent binding reagents is capable of specifically binding to atleast one of the one or more cellular component targets. The sequencesof the binding reagent oligonucleotides associated with the two or morecellular component binding reagents can be identical. The sequences ofthe binding reagent oligonucleotides associated with the two or morecellular component binding reagents can comprise different sequences.Each of the plurality of compositions can comprise the two or morecellular component binding reagents.

The number of different types of cellular component binding reagents(e.g., a CD147 antibody and a CD47 antibody) in a composition can bedifferent in different implementations. A composition with two or moredifferent types of cellular component binding reagents can be referredto herein as a cellular component binding reagent cocktail (e.g., asample indexing composition cocktail). The number of different types ofcellular component binding reagents in a cocktail can vary. In someembodiments, the number of different types of cellular component bindingreagents in cocktail can be, or be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 10000, 100000, or a number or a range between any two of thesevalues. In some embodiments, the number of different types of cellularcomponent binding reagents in cocktail can be at least, or be at most,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 10000, or 100000. The differenttypes of cellular component binding reagents can be conjugated tobinding reagent oligonucleotides with the same or different sequences(e.g., sample indexing sequences).

Methods of Quantitative Analysis of Cellular Component Targets

In some embodiments, the methods disclosed herein can also be used forquantitative analysis of a plurality of cellular component targets (forexample, protein targets) in a sample using the compositions disclosedherein and oligonucleotide probes that can associate a barcode sequence(e.g., a molecular label sequence) to the oligonucleotides of thecellular component binding reagents (e.g., protein binding reagents).The oligonucleotides of the cellular component binding reagents can be,or comprise, an antibody oligonucleotide, a sample indexingoligonucleotide, a cell identification oligonucleotide, a controlparticle oligonucleotide, a control oligonucleotide, an interactiondetermination oligonucleotide, etc. In some embodiments, the sample canbe a single cell, a plurality of cells, a tissue sample, a tumor sample,a blood sample, or the like. In some embodiments, the sample cancomprise a mixture of cell types, such as normal cells, tumor cells,blood cells, B cells, T cells, maternal cells, fetal cells, etc., or amixture of cells from different subjects.

In some embodiments, the sample can comprise a plurality of single cellsseparated into individual compartments, such as microwells in amicrowell array.

In some embodiments, the binding target of the plurality of cellularcomponent target (i.e., the cellular component target) can be, orcomprise, a carbohydrate, a lipid, a protein, an extracellular protein,a cell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, an intracellular protein, or any combinationthereof. In some embodiments, the cellular component target is a proteintarget. In some embodiments, the plurality of cellular component targetscomprises a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, an antibody, a major histocompatibility complex, atumor antigen, a receptor, or any combination thereof. In someembodiments, the plurality of cellular component targets can compriseintracellular cellular components. In some embodiments, the plurality ofcellular components can be at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, at least 99%, or more, of all the encoded cellularcomponents in an organism. In some embodiments, the plurality ofcellular component targets can comprise at least 2, at least 3, at least4, at least 5, at least 10, at least 20, at least 30, at least 40, atleast 50, at least 100, at least 1000, at least 10000, or more differentcellular component targets.

In some embodiments, the plurality of cellular component bindingreagents is contacted with the sample for specific binding with theplurality of cellular component targets. Unbound cellular componentbinding reagents can be removed, for example, by washing. In embodimentswhere the sample comprises cells, any cellular component bindingreagents not specifically bound to the cells can be removed.

In some instances, cells from a population of cells can be separated(e.g., isolated) into wells of a substrate of the disclosure. Thepopulation of cells can be diluted prior to separating. The populationof cells can be diluted such that at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or100%, of wells of the substrate receive a single cell. The population ofcells can be diluted such that at most 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%, of wells of the substrate receive a single cell. The population ofcells can be diluted such that the number of cells in the dilutedpopulation is, or is at least, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, ofthe number of wells on the substrate. The population of cells can bediluted such that the number of cells in the diluted population is, oris at least, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the number of wellson the substrate. In some instances, the population of cells is dilutedsuch that the number of cell is about 10% of the number of wells in thesubstrate.

Distribution of single cells into wells of the substrate can follow aPoisson distribution. For example, there can be at least a 0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or more probability that awell of the substrate has more than one cell. There can be at least a0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or moreprobability that a well of the substrate has more than one cell.Distribution of single cells into wells of the substrate can be random.Distribution of single cells into wells of the substrate can benon-random. The cells can be separated such that a well of the substratereceives only one cell.

In some embodiments, the cellular component binding reagents can beadditionally conjugated with fluorescent molecules to enable flowsorting of cells into individual compartments.

In some embodiments, the methods disclosed herein provide contacting aplurality of compositions with the sample for specific binding with theplurality of cellular component targets. It would be appreciated thatthe conditions used may allow specific binding of the cellular componentbinding reagents, e.g., antibodies, to the cellular component targets.Following the contacting step, unbound compositions can be removed. Forexample, in embodiments where the sample comprises cells, and thecompositions specifically bind to cellular component targets arecell-surface cellular components, such as cell-surface proteins, unboundcompositions can be removed by washing the cells with buffer such thatonly compositions that specifically bind to the cellular componenttargets remain with the cells.

In some embodiments, the methods disclosed herein can compriseassociating an oligonucleotide (e.g., a barcode, or a stochasticbarcode), including a barcode sequence (such as a molecular label), acell label, a sample label, etc., or any combination thereof, to theplurality of oligonucleotides associated with the cellular componentbinding reagents. For example, a plurality of oligonucleotide probescomprising a barcode can be used to hybridize to the plurality ofoligonucleotides of the compositions.

In some embodiments, the plurality of oligonucleotide probes can beimmobilized on solid supports. The solid supports can be free floating,e.g., beads in a solution. The solid supports can be embedded in asemi-solid or solid array. In some embodiments, the plurality ofoligonucleotide probes may not be immobilized on solid supports. Whenthe plurality of oligonucleotide probes are in close proximity to theplurality associated with oligonucleotides of the cellular componentbinding reagents, the plurality of oligonucleotides of the cellularcomponent binding reagents can hybridize to the oligonucleotide probes.The oligonucleotide probes can be contacted at a non-depletable ratiosuch that each distinct oligonucleotide of the cellular componentbinding reagents can associate with oligonucleotide probes havingdifferent barcode sequences (e.g., molecular labels) of the disclosure.

In some embodiments, the methods disclosed herein provide detaching theoligonucleotides from the cellular component binding reagents that arespecifically bound to the cellular component targets. Detachment can beperformed in a variety of ways to separate the chemical group from thecellular component binding reagent, such as UV photocleaving, chemicaltreatment (e.g., dithiothreitol treatment), heating, enzyme treatment,or any combination thereof. Detaching the oligonucleotide from thecellular component binding reagent can be performed either before,after, or during the step of hybridizing the plurality ofoligonucleotide probes to the plurality of oligonucleotides of thecompositions.

Methods of Simultaneous Quantitative Analysis of Cellular Component andNucleic Acid Targets

In some embodiments, the methods disclosed herein can also be used forsimultaneous quantitative analysis of a plurality of cellular componenttargets (e.g., protein targets) and a plurality of nucleic acid targetmolecules in a sample using the compositions disclosed herein andoligonucleotide probes that can associate a barcode sequence (e.g., amolecular label sequence) to both the oligonucleotides of the cellularcomponent binding reagents and nucleic acid target molecules. Othermethods of simultaneous quantitative analysis of a plurality of cellularcomponent targets and a plurality of nucleic acid target molecules aredescribed in U.S. patent application Ser. No. 15/715,028, filed on Sep.25, 2017; the content of which is incorporated herein by reference inits entirety. In some embodiments, the sample can be a single cell, aplurality of cells, a tissue sample, a tumor sample, a blood sample, orthe like. In some embodiments, the sample can comprise a mixture of celltypes, such as normal cells, tumor cells, blood cells, B cells, T cells,maternal cells, fetal cells, or a mixture of cells from differentsubjects.

In some embodiments, the sample can comprise a plurality of single cellsseparated into individual compartments, such as microwells in amicrowell array.

In some embodiments, the plurality of cellular component targetscomprises a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, an antibody, a major histocompatibility complex, atumor antigen, a receptor, or any combination thereof. In someembodiments, the plurality of cellular component targets can compriseintracellular cellular components. In some embodiments, the plurality ofcellular components can be, or be about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or anumber or a range between any two of these values, of all the cellularcomponents, such as expressed proteins, in an organism, or one or morecell of the organism. In some embodiments, the plurality of cellularcomponents can be at least, or be at most, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%,of all the cellular components, such as proteins could be expressed, inan organism, or one or more cells of the organism. In some embodiments,the plurality of cellular component targets can comprise, or compriseabout, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, 10000, or a number ora range between any two of these values, different cellular componenttargets. In some embodiments, the plurality of cellular componenttargets can comprise at least, or comprise at most, 2, 3, 4, 5, 10, 20,30, 40, 50, 100, 1000, or 10000, different cellular component targets.

In some embodiments, the plurality of cellular component bindingreagents is contacted with the sample for specific binding with theplurality of cellular component targets. Unbound cellular componentbinding reagents can be removed, for example, by washing. In embodimentswhere the sample comprises cells, any cellular component bindingreagents not specifically bound to the cells can be removed.

In some instances, cells from a population of cells can be separated(e.g., isolated) into wells of a substrate of the disclosure. Thepopulation of cells can be diluted prior to separating. The populationof cells can be diluted such that at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% of wells of the substrate receive a single cell. The population ofcells can be diluted such that at most 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of wells of the substrate receive a single cell. The population of cellscan be diluted such that the number of cells in the diluted populationis, or is at least, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the number ofwells on the substrate. The population of cells can be diluted such thatthe number of cells in the diluted population is, or is at least, 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of the number of wells on thesubstrate. In some instances, the population of cells is diluted suchthat the number of cell is about 10% of the number of wells in thesubstrate.

Distribution of single cells into wells of the substrate can follow aPoisson distribution. For example, there can be at least a 0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or more probability that awell of the substrate has more than one cell. There can be at least a0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or moreprobability that a well of the substrate has more than one cell.Distribution of single cells into wells of the substrate can be random.Distribution of single cells into wells of the substrate can benon-random. The cells can be separated such that a well of the substratereceives only one cell.

In some embodiments, the cellular component binding reagents can beadditionally conjugated with fluorescent molecules to enable flowsorting of cells into individual compartments.

In some embodiments, the methods disclosed herein provide contacting aplurality of compositions with the sample for specific binding with theplurality of cellular component targets. It would be appreciated thatthe conditions used may allow specific binding of the cellular componentbinding reagents, e.g., antibodies, to the cellular component targets.Following the contacting step, unbound compositions can be removed. Forexample, in embodiments where the sample comprises cells, and thecompositions specifically bind to cellular component targets are on thecell surface, such as cell-surface proteins, unbound compositions can beremoved by washing the cells with buffer such that only compositionsthat specifically bind to the cellular component targets remain with thecells.

In some embodiments, the methods disclosed herein can provide releasingthe plurality of nucleic acid target molecules from the sample, e.g.,cells. For example, the cells can be lysed to release the plurality ofnucleic acid target molecules. Cell lysis may be accomplished by any ofa variety of means, for example, by chemical treatment, osmotic shock,thermal treatment, mechanical treatment, optical treatment, or anycombination thereof. Cells may be lysed by addition of a cell lysisbuffer comprising a detergent (e.g., SDS, Li dodecyl sulfate, TritonX-100, Tween-20, or NP-40), an organic solvent (e.g., methanol oracetone), or digestive enzymes (e.g., proteinase K, pepsin, or trypsin),or any combination thereof.

It would be appreciated by one of ordinary skill in the art that theplurality of nucleic acid molecules can comprise a variety of nucleicacid molecules. In some embodiments, the plurality of nucleic acidmolecules can comprise, DNA molecules, RNA molecules, genomic DNAmolecules, mRNA molecules, rRNA molecules, siRNA molecules, or acombination thereof, and can be double-stranded or single-stranded. Insome embodiments, the plurality of nucleic acid molecules comprise, orcomprise about, 100, 1000, 10000, 20000, 30000, 40000, 50000, 100000,1000000, or a number or a range between any two of these values,species. In some embodiments, the plurality of nucleic acid moleculescomprise at least, or comprise at most, 100, 1000, 10000, 20000, 30000,40000, 50000, 100000, or 1000000, species. In some embodiments, theplurality of nucleic acid molecules can be from a sample, such as asingle cell, or a plurality of cells. In some embodiments, the pluralityof nucleic acid molecules can be pooled from a plurality of samples,such as a plurality of single cells.

In some embodiments, the methods disclosed herein can compriseassociating a barcode (e.g., a stochastic barcode), which can include abarcode sequence (such as a molecular label), a cell label, a samplelabel, etc., or any combination thereof, to the plurality of nucleicacid target molecules and the plurality of oligonucleotides of thecellular component binding reagents. For example, a plurality ofoligonucleotide probes comprising a stochastic barcode can be used tohybridize to the plurality of nucleic acid target molecules and theplurality of oligonucleotides of the compositions.

In some embodiments, the plurality of oligonucleotide probes can beimmobilized on solid supports. The solid supports can be free floating,e.g., beads in a solution. The solid supports can be embedded in asemi-solid or solid array. In some embodiments, the plurality ofoligonucleotide probes may not be immobilized on solid supports. Whenthe plurality of oligonucleotide probes are in close proximity to theplurality of nucleic acid target molecules and the plurality ofoligonucleotides of the cellular component binding reagents, theplurality of nucleic acid target molecules and the plurality ofoligonucleotides of the cellular component binding reagents canhybridize to the oligonucleotide probes. The oligonucleotide probes canbe contacted at a non-depletable ratio such that each distinct nucleicacid target molecules and oligonucleotides of the cellular componentbinding reagents can associate with oligonucleotide probes havingdifferent barcode sequences (e.g., molecular labels) of the disclosure.

In some embodiments, the methods disclosed herein provide detaching theoligonucleotides from the cellular component binding reagents that arespecifically bound to the cellular component targets. Detachment can beperformed in a variety of ways to separate the chemical group from thecellular component binding reagent, such as UV photocleaving, chemicaltreatment (e.g., dithiothreitol treatment), heating, enzyme treatment,or any combination thereof. Detaching the oligonucleotide from thecellular component binding reagent can be performed either before,after, or during the step of hybridizing the plurality ofoligonucleotide probes to the plurality of nucleic acid target moleculesand the plurality of oligonucleotides of the compositions.

Simultaneous Quantitative Analysis of Protein and Nucleic Acid Targets

In some embodiments, the methods disclosed herein also can be used forsimultaneous quantitative analysis of multiple types of targetmolecules, for example protein and nucleic acid targets. For example,the target molecules can be, or comprise, cellular components. FIG. 6shows a schematic illustration of an exemplary method of simultaneousquantitative analysis of both nucleic acid targets and other cellularcomponent targets (e.g., proteins) in single cells. In some embodiments,a plurality of compositions 605, 605 b, 605 c, etc., each comprising acellular component binding reagent, such as an antibody, is provided.Different cellular component binding reagents, such as antibodies, whichbind to different cellular component targets are conjugated withdifferent unique identifiers. Next, the cellular component bindingreagents can be incubates with a sample containing a plurality of cells610. The different cellular component binding reagents can specificallybind to cellular components on the cell surface, such as a cell marker,a B-cell receptor, a T-cell receptor, an antibody, a majorhistocompatibility complex, a tumor antigen, a receptor, or anycombination thereof. Unbound cellular component binding reagents can beremoved, e.g., by washing the cells with a buffer. The cells with thecellular component binding reagents can be then separated into aplurality of compartments, such as a microwell array, wherein a singlecompartment 615 is sized to fit a single cell and a single bead 620.Each bead can comprise a plurality of oligonucleotide probes, which cancomprise a cell label that is common to all oligonucleotide probes on abead, and barcode sequences (e.g., molecular label sequences). In someembodiments, each oligonucleotide probe can comprise a target bindingregion, for example, a poly(dT) sequence. The oligonucleotides 625conjugated to the cellular component binding reagent can be detachedfrom the cellular component binding reagent using chemical, optical orother means. The cell can be lysed 635 to release nucleic acids withinthe cell, such as genomic DNA or cellular mRNA 630. Cellular mRNA 630,oligonucleotides 625 or both can be captured by the oligonucleotideprobes on bead 620, for example, by hybridizing to the poly(dT)sequence. A reverse transcriptase can be used to extend theoligonucleotide probes hybridized to the cellular mRNA 630 and theoligonucleotides 625 using the cellular mRNA 630 and theoligonucleotides 625 as templates. The extension products produced bythe reverse transcriptase can be subject to amplification andsequencing. Sequencing reads can be subject to demultiplexing ofsequences or identifies of cell labels, barcodes (e.g., molecularlabels), genes, cellular component binding reagent specificoligonucleotides (e.g., antibody specific oligonucleotides), etc., whichcan give rise to a digital representation of cellular components andgene expression of each single cell in the sample.

Association of Barcodes

The oligonucleotides associated with the cellular component bindingreagents (e.g., antigen binding reagents or protein binding reagents)and/or the nucleic acid molecules may randomly associate with theoligonucleotide probes. The oligonucleotides associated with thecellular component binding reagents, referred to herein as bindingreagent oligonucleotides, can be, or comprise oligonucleotides of thedisclosure, such as an antibody oligonucleotide, a sample indexingoligonucleotide, a cell identification oligonucleotide, a controlparticle oligonucleotide, a control oligonucleotide, an interactiondetermination oligonucleotide, etc. Association can, for example,comprise hybridization of an oligonucleotide probe's target bindingregion to a complementary portion of the target nucleic acid moleculeand/or the oligonucleotides of the protein binding reagents. Forexample, a oligo(dT) region of a barcode (e.g., a stochastic barcode)can interact with a poly(A) tail of a target nucleic acid moleculeand/or a poly(A) tail of an oligonucleotide of a protein bindingreagent. The assay conditions used for hybridization (e.g., buffer pH,ionic strength, temperature, etc.) can be chosen to promote formation ofspecific, stable hybrids.

The disclosure provides for methods of associating a molecular labelwith a target nucleic acid and/or an oligonucleotide associated with acellular component binding reagent using reverse transcription. As areverse transcriptase can use both RNA and DNA as template. For example,the oligonucleotide originally conjugated on the cellular componentbinding reagent can be either RNA or DNA bases, or both. A bindingreagent oligonucleotide can be copied and linked (e.g., covalentlylinked) to a cell label and a barcode sequence (e.g., a molecular label)in addition to the sequence, or a portion thereof, of the bindingreagent sequence. As another example, an mRNA molecule can be copied andlinked (e.g., covalently linked) to a cell label and a barcode sequence(e.g., a molecular label) in addition to the sequence of the mRNAmolecule, or a portion thereof.

In some embodiments, molecular labels can be added by ligation of anoligonucleotide probe target binding region and a portion of the targetnucleic acid molecule and/or the oligonucleotides associated with (e.g.,currently, or previously, associated with) with cellular componentbinding reagents. For example, the target binding region may comprise anucleic acid sequence that can be capable of specific hybridization to arestriction site overhang (e.g., an EcoRI sticky-end overhang). Themethods can further comprise treating the target nucleic acids and/orthe oligonucleotides associated with cellular component binding reagentswith a restriction enzyme (e.g., EcoRI) to create a restriction siteoverhang. A ligase (e.g., T4 DNA ligase) may be used to join the twofragments.

Determining the Number or Presence of Unique Molecular Label Sequences

In some embodiments, the methods disclosed herein comprise determiningthe number or presence of unique molecular label sequences for eachunique identifier, each nucleic acid target molecule, and/or eachbinding reagent oligonucleotides (e.g., antibody oligonucleotides). Forexample, the sequencing reads can be used to determine the number ofunique molecular label sequences for each unique identifier, eachnucleic acid target molecule, and/or each binding reagentoligonucleotide. As another example, the sequencing reads can be used todetermine the presence or absence of a molecular label sequence (such asa molecular label sequence associated with a target, a binding reagentoligonucleotide, an antibody oligonucleotide, a sample indexingoligonucleotide, a cell identification oligonucleotide, a controlparticle oligonucleotide, a control oligonucleotide, an interactiondetermination oligonucleotide, etc. in the sequencing reads).

In some embodiments, the number of unique molecular label sequences foreach unique identifier, each nucleic acid target molecule, and/or eachbinding reagent oligonucleotide indicates the quantity of each cellularcomponent target (e.g., an antigen target or a protein target) and/oreach nucleic acid target molecule in the sample. In some embodiments,the quantity of a cellular component target and the quantity of itscorresponding nucleic acid target molecules, e.g., mRNA molecules, canbe compared to each other. In some embodiments, the ratio of thequantity of a cellular component target and the quantity of itscorresponding nucleic acid target molecules, e.g., mRNA molecules, canbe calculated. The cellular component targets can be, for example, cellsurface protein markers. In some embodiments, the ratio between theprotein level of a cell surface protein marker and the level of the mRNAof the cell surface protein marker is low.

The methods disclosed herein can be used for a variety of applications.For example, the methods disclosed herein can be used for proteomeand/or transcriptome analysis of a sample. In some embodiments, themethods disclosed herein can be used to identify a cellular componenttarget and/or a nucleic acid target, i.e., a biomarker, in a sample. Insome embodiments, the cellular component target and the nucleic acidtarget correspond to each other, i.e., the nucleic acid target encodesthe cellular component target. In some embodiments, the methodsdisclosed herein can be used to identify cellular component targets thathave a desired ratio between the quantity of the cellular componenttarget and the quantity of its corresponding nucleic acid targetmolecule in a sample, e.g., mRNA molecule. In some embodiments, theratio is, or is about, 0.001, 0.01, 0.1, 1, 10, 100, 1000, or a numberor a range between any two of the above values. In some embodiments, theratio is at least, or is at most, 0.001, 0.01, 0.1, 1, 10, 100, or 1000.In some embodiments, the methods disclosed herein can be used toidentify cellular component targets in a sample that the quantity of itscorresponding nucleic acid target molecule in the sample is, or isabout, 1000, 100, 10, 5, 2 1, 0, or a number or a range between any twoof these values. In some embodiments, the methods disclosed herein canbe used to identify cellular component targets in a sample that thequantity of its corresponding nucleic acid target molecule in the sampleis more than, or less than, 1000, 100, 10, 5, 2, 1, or 0.

Compositions and Kits

Some embodiments disclosed herein provide kits and compositions forsimultaneous quantitative analysis of a plurality of cellular components(e.g., proteins) and/or a plurality of nucleic acid target molecules ina sample. The kits and compositions can, in some embodiments, comprise aplurality of cellular component binding reagents (e.g., a plurality ofprotein binding reagents) each conjugated with an oligonucleotide,wherein the oligonucleotide comprises a unique identifier for thecellular component binding reagent, and a plurality of oligonucleotideprobes, wherein each of the plurality of oligonucleotide probescomprises a target binding region, a barcode sequence (e.g., a molecularlabel sequence), wherein the barcode sequence is from a diverse set ofunique barcode sequences. In some embodiments, each of theoligonucleotides can comprise a molecular label, a cell label, a samplelabel, or any combination thereof. In some embodiments, each of theoligonucleotides can comprise a linker. In some embodiments, each of theoligonucleotides can comprise a binding site for an oligonucleotideprobe, such as a poly(A) tail. For example, the poly(A) tail can be,e.g., oligodA₁₈ (unanchored to a solid support) or oligoA₁₈V (anchoredto a solid support). The oligonucleotides can comprise DNA residues, RNAresidues, or both.

Disclosed herein includes a plurality of sample indexing compositions.Each of the plurality of sample indexing compositions can comprise twoor more cellular component binding reagents. Each of the two or morecellular component binding reagents can be associated with a sampleindexing oligonucleotide. At least one of the two or more cellularcomponent binding reagents can be capable of specifically binding to atleast one cellular component target. The sample indexing oligonucleotidecan comprise a sample indexing sequence for identifying sample origin ofone or more cells of a sample. Sample indexing sequences of at least twosample indexing compositions of the plurality of sample indexingcompositions can comprise different sequences.

Disclosed herein include kits comprising control particle compositions.In some embodiments, the control particle composition comprises aplurality of control particle oligonucleotides associated with a controlparticle, wherein each of the plurality of control particleoligonucleotides comprises a control barcode sequence and a poly(dA)region. At least two of the plurality of control particleoligonucleotides can comprise different control barcode sequences. Thecontrol particle oligonucleotide can comprise a molecular labelsequence. The control particle oligonucleotide can comprise a bindingsite for a universal primer. Also disclosed herein include kits forsequencing control. In some embodiments, the kit comprises: a controlparticle composition comprising a plurality of control particleoligonucleotides associated with a control particle, wherein each of theplurality of control particle oligonucleotides comprises a controlbarcode sequence and a poly(dA) region.

Disclosed herein include kits comprising a plurality of controlcompositions for sequencing control. In some embodiments, each of theplurality of control compositions comprises a cellular component bindingreagent associated with a control oligonucleotide, wherein the cellularcomponent binding reagent is capable of specifically binding to at leastone of a plurality of binding targets, and wherein the controloligonucleotide comprises a control barcode sequence and a pseudo-targetregion comprising a sequence substantially complementary to thetarget-binding region of at least one of the plurality of barcodes. Insome embodiments, the pseudo-target region comprises a poly(dA) region.

Disclosed herein include kits comprising sample indexing compositionsfor cell identification. In some embodiments. Each of two sampleindexing compositions comprises a cellular component binding reagent(e.g., a protein binding reagent) associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of one or more cellularcomponent targets (e.g., one or more protein targets), wherein thesample indexing oligonucleotide comprises a sample indexing sequence,and wherein sample indexing sequences of the two sample indexingcompositions comprise different sequences. In some embodiments, thesample indexing oligonucleotide comprises a molecular label sequence, abinding site for a universal primer, or a combination thereof.

Disclosed herein includes kits for cell identification. In someembodiments, the kit comprises: two or more sample indexingcompositions. Each of the two or more sample indexing compositions cancomprise a cellular component binding reagent (e.g., an antigen bindingreagent) associated with a sample indexing oligonucleotide, wherein thecellular component binding reagent is capable of specifically binding toat least one of one or more cellular component targets, wherein thesample indexing oligonucleotide comprises a sample indexing sequence,and wherein sample indexing sequences of the two sample indexingcompositions comprise different sequences. In some embodiments, thesample indexing oligonucleotide comprises a molecular label sequence, abinding site for a universal primer, or a combination thereof. Disclosedherein includes kits for multiplet identification. In some embodiments,the kit comprises two sample indexing compositions. Each of two sampleindexing compositions can comprise a cellular component binding reagent(e.g., an antigen binding reagent) associated with a sample indexingoligonucleotide, wherein the antigen binding reagent is capable ofspecifically binding to at least one of one or more cellular componenttargets (e.g., antigen targets), wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of the two sample indexing compositions comprisedifferent sequences.

Disclosed herein include kits for identifying interactions betweencellular components, for example protein-protein interactions. In someembodiments, the kit comprises: a first pair of interactiondetermination compositions, wherein each of the first pair ofinteraction determination compositions comprises a protein bindingreagent associated with an interaction determination oligonucleotide,wherein the protein binding reagent of one of the first pair ofinteraction determination compositions is capable of specificallybinding to a first protein target and a protein binding reagent of theother of the first pair of interaction determination compositions iscapable of specifically binding to the second protein target, whereinthe interaction determination oligonucleotide comprises an interactiondetermination sequence and a bridge oligonucleotide hybridizationregion, and wherein the interaction determination sequences of the firstpair of interaction determination compositions comprise differentsequences; and a plurality of bridge oligonucleotides each comprisingtwo hybridization regions capable of specifically binding to the bridgeoligonucleotide hybridization regions of the first pair of interactiondetermination compositions.

The unique identifiers (or oligonucleotides associated with cellularcomponent binding reagents, such as binding reagent oligonucleotides,antibody oligonucleotides, sample indexing oligonucleotides, cellidentification oligonucleotides, control particle oligonucleotides,control oligonucleotides, or interaction determination oligonucleotides)can have any suitable length, for example, from about 25 nucleotides toabout 45 nucleotides long. In some embodiments, the unique identifiercan have a length that is, is about, is less than, is greater than, 4nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides,9 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 70nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 200nucleotides, or a range that is between any two of the above values.

In some embodiments, the unique identifiers are selected from a diverseset of unique identifiers. The diverse set of unique identifiers cancomprise, or comprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or arange between any two of these values, different unique identifiers. Thediverse set of unique identifiers can comprise at least, or comprise atmost, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, or 5000, different unique identifiers. In someembodiments, the set of unique identifiers is designed to have minimalsequence homology to the DNA or RNA sequences of the sample to beanalyzed. In some embodiments, the sequences of the set of uniqueidentifiers are different from each other, or the complement thereof,by, or by about, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides,9 nucleotides, 10 nucleotides, or a number or a range between any two ofthese values. In some embodiments, the sequences of the set of uniqueidentifiers are different from each other, or the complement thereof, byat least, or by at most, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides,9 nucleotides, or 10 nucleotides.

In some embodiments, the unique identifiers can comprise a binding sitefor a primer, such as universal primer. In some embodiments, the uniqueidentifiers can comprise at least two binding sites for a primer, suchas a universal primer. In some embodiments, the unique identifiers cancomprise at least three binding sites for a primer, such as a universalprimer. The primers can be used for amplification of the uniqueidentifiers, for example, by PCR amplification. In some embodiments, theprimers can be used for nested PCR reactions.

Any suitable cellular component binding reagents are contemplated inthis disclosure, such as any protein binding reagents (e.g., antibodiesor fragments thereof, aptamers, small molecules, ligands, peptides,oligonucleotides, etc., or any combination thereof). In someembodiments, the cellular component binding reagents can be polyclonalantibodies, monoclonal antibodies, recombinant antibodies, single-chainantibody (scAb), or fragments thereof, such as Fab, Fv, etc. In someembodiments, the plurality of protein binding reagents can comprise, orcomprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000, 5000, or a number or a range between anytow of these values, different protein binding reagents. In someembodiments, the plurality of protein binding reagents can comprise atleast, or comprise at most, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 5000, differentprotein binding reagents.

In some embodiments, the oligonucleotide is conjugated with the cellularcomponent binding reagent through a linker. In some embodiments, theoligonucleotide can be conjugated with the protein binding reagentcovalently. In some embodiment, the oligonucleotide can be conjugatedwith the protein binding reagent non-covalently. In some embodiments,the linker can comprise a chemical group that reversibly or irreversbilyattached the oligonucleotide to the protein binding reagents. Thechemical group can be conjugated to the linker, for example, through anamine group. In some embodiments, the linker can comprise a chemicalgroup that forms a stable bond with another chemical group conjugated tothe protein binding reagent. For example, the chemical group can be a UVphotocleavable group, a disulfide bond, a streptavidin, a biotin, anamine, etc. In some embodiments, the chemical group can be conjugated tothe protein binding reagent through a primary amine on an amino acid,such as lysine, or the N-terminus. The oligonucleotide can be conjugatedto any suitable site of the protein binding reagent, as long as it doesnot interfere with the specific binding between the protein bindingreagent and its protein target. In embodiments where the protein bindingreagent is an antibody, the oligonucleotide can be conjugated to theantibody anywhere other than the antigen-binding site, for example, theFc region, the C_(H)1 domain, the C_(H)2 domain, the C_(H)3 domain, theC_(L) domain, etc. In some embodiments, each protein binding reagent canbe conjugated with a single oligonucleotide molecule. In someembodiments, each protein binding reagent can be conjugated with, orwith about, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, or a number or arange between any tow of these values, oligonucleotide molecules,wherein each of the oligonucleotide molecule comprises the same uniqueidentifier. In some embodiments, each protein binding reagent can beconjugated with more than one oligonucleotide molecule, for example, atleast, or at most, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, or 1000,oligonucleotide molecules, wherein each of the oligonucleotide moleculecomprises the same unique identifier.

In some embodiments, the plurality of cellular component bindingreagents (e.g., protein binding reagents) are capable of specificallybinding to a plurality of cellular component targets (e.g., proteintargets) in a sample. The sample can be, or comprise, a single cell, aplurality of cells, a tissue sample, a tumor sample, a blood sample, orthe like. In some embodiments, the plurality of cellular componenttargets comprises a cell-surface protein, a cell marker, a B-cellreceptor, a T-cell receptor, an antibody, a major histocompatibilitycomplex, a tumor antigen, a receptor, or any combination thereof. Insome embodiments, the plurality of cellular component targets cancomprise intracellular proteins. In some embodiments, the plurality ofcellular component targets can comprise intracellular proteins. In someembodiments, the plurality of cellular component targets can be, or beabout, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or a number or a range between any two ofthese values of all cellular component targets (e.g., proteins expressedor could be expressed) in an organism. In some embodiments, theplurality of cellular component targets can be at least, or be at most,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 98%, or 99%, of all cellular component targets (e.g.,proteins expressed or could be expressed) in an organism. In someembodiments, the plurality of cellular component targets can comprise,or comprise about, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, 10000, ora number or a range between any two of these values, different cellularcomponent targets. In some embodiments, the plurality of cellularcomponent targets can comprise at least, or comprise at most, 2, 3, 4,5, 10, 20, 30, 40, 50, 100, 1000, or 10000, different cellular componenttargets.

Sample Indexing Using Oligonucleotide-Conjugated Cellular ComponentBinding Reagent

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more cellular component targets, whereineach of the plurality of sample indexing compositions comprises acellular component binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; removingunbound sample indexing compositions of the plurality of sample indexingcompositions; barcoding (e.g., stochastically barcoding) the sampleindexing oligonucleotides using a plurality of barcodes (e.g.,stochastic barcodes) to create a plurality of barcoded sample indexingoligonucleotides; obtaining sequencing data of the plurality of barcodedsample indexing oligonucleotides; and identifying sample origin of atleast one cell of the one or more cells based on the sample indexingsequence of at least one barcoded sample indexing oligonucleotide of theplurality of barcoded sample indexing oligonucleotides.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

An oligonucleotide-conjugated with an antibody, an oligonucleotide forconjugation with an antibody, or an oligonucleotide previouslyconjugated with an antibody is referred to herein as an antibodyoligonucleotide (“AbOligo”). Antibody oligonucleotides in the context ofsample indexing are referred to herein as sample indexingoligonucleotides. An antibody conjugated with an antibodyoligonucleotide is referred to herein as a hot antibody or anoligonucleotide antibody. An antibody not conjugated with an antibodyoligonucleotide is referred to herein as a cold antibody or anoligonucleotide free antibody. An oligonucleotide-conjugated with abinding reagent (e.g., a protein binding reagent), an oligonucleotidefor conjugation with a binding reagent, or an oligonucleotide previouslyconjugated with a binding reagent is referred to herein as a reagentoligonucleotide. Reagent oligonucleotides in the context of sampleindexing are referred to herein as sample indexing oligonucleotides. Abinding reagent conjugated with an antibody oligonucleotide is referredto herein as a hot binding reagent or an oligonucleotide bindingreagent. A binding reagent not conjugated with an antibodyoligonucleotide is referred to herein as a cold binding reagent or anoligonucleotide free binding reagent.

FIG. 7 shows a schematic illustration of an exemplary workflow usingoligonucleotide-conjugated cellular component binding reagents forsample indexing. In some embodiments, a plurality of compositions 705 a,705 b, etc., each comprising a binding reagent is provided. The bindingreagent can be a protein binding reagent, such as an antibody. Thecellular component binding reagent can comprise an antibody, a tetramer,an aptamers, a protein scaffold, or a combination thereof. The bindingreagents of the plurality of compositions 705 a, 705 b can bind to anidentical cellular component target. For example, the binding reagentsof the plurality of compositions 705, 705 b can be identical (except forthe sample indexing oligonucleotides associated with the bindingreagents).

Different compositions can include binding reagents conjugated withsample indexing oligonucleotides with different sample indexingsequences. The number of different compositions can be different indifferent implementations. In some embodiments, the number of differentcompositions can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or a numberor a range between any two of these values. In some embodiments, thenumber of different compositions can be at least, or be at most, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, or 10000.

In some embodiments, the sample indexing oligonucleotides of bindingreagents in one composition can include an identical sample indexingsequence. The sample indexing oligonucleotides of binding reagents inone composition may not be identical. In some embodiments, thepercentage of sample indexing oligonucleotides of binding reagents inone composition with an identical sample indexing sequence can be, or beabout, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or a number or arange between any two of these values. In some embodiments, thepercentage of sample indexing oligonucleotides of binding reagents inone composition with an identical sample indexing sequence can be atleast, or be at most, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%.

The compositions 705 a and 705 b can be used to label samples ofdifferent samples. For example, the sample indexing oligonucleotides ofthe cellular component binding reagent in the composition 705 a can haveone sample indexing sequence and can be used to label cells 710 a, shownas black circles, in a sample 707 a, such as a sample of a patient. Thesample indexing oligonucleotides of the cellular component bindingreagents in the composition 705 b can have another sample indexingsequence and can be used to label cells 710 b, shown as hatched circles,in a sample 707 b, such as a sample of another patient or another sampleof the same patient. The cellular component binding reagents canspecifically bind to cellular component targets or proteins on the cellsurface, such as a cell marker, a B-cell receptor, a T-cell receptor, anantibody, a major histocompatibility complex, a tumor antigen, areceptor, or any combination thereof. Unbound cellular component bindingreagents can be removed, e.g., by washing the cells with a buffer.

The cells with the cellular component binding reagents can be thenseparated into a plurality of compartments, such as a microwell array,wherein a single compartment 715 a, 715 b is sized to fit a single cell710 a and a single bead 720 a or a single cell 710 b and a single bead720 b. Each bead 720 a, 720 b can comprise a plurality ofoligonucleotide probes, which can comprise a cell label that is commonto all oligonucleotide probes on a bead, and molecular label sequences.In some embodiments, each oligonucleotide probe can comprise a targetbinding region, for example, a poly(dT) sequence. The sample indexingoligonucleotides 725 a conjugated to the cellular component bindingreagent of the composition 705 a can be configured to be detachable ornon-detachable from the cellular component binding reagent. The sampleindexing oligonucleotides 725 a conjugated to the cellular componentbinding reagent of the composition 705 a can be detached from thecellular component binding reagent using chemical, optical or othermeans. The sample indexing oligonucleotides 725 b conjugated to thecellular component binding reagent of the composition 705 b can beconfigured to be detachable or non-detachable from the cellularcomponent binding reagent. The sample indexing oligonucleotides 725 bconjugated to the cellular component binding reagent of the composition705 b can be detached from the cellular component binding reagent usingchemical, optical or other means.

The cell 710 a can be lysed to release nucleic acids within the cell 710a, such as genomic DNA or cellular mRNA 730 a. The lysed cell 735 a isshown as a dotted circle. Cellular mRNA 730 a, sample indexingoligonucleotides 725 a, or both can be captured by the oligonucleotideprobes on bead 720 a, for example, by hybridizing to the poly(dT)sequence. A reverse transcriptase can be used to extend theoligonucleotide probes hybridized to the cellular mRNA 730 a and theoligonucleotides 725 a using the cellular mRNA 730 a and theoligonucleotides 725 a as templates. The extension products produced bythe reverse transcriptase can be subject to amplification andsequencing.

Similarly, the cell 710 b can be lysed to release nucleic acids withinthe cell 710 b, such as genomic DNA or cellular mRNA 730 b. The lysedcell 735 b is shown as a dotted circle. Cellular mRNA 730 b, sampleindexing oligonucleotides 725 b, or both can be captured by theoligonucleotide probes on bead 720 b, for example, by hybridizing to thepoly(dT) sequence. A reverse transcriptase can be used to extend theoligonucleotide probes hybridized to the cellular mRNA 730 b and theoligonucleotides 725 b using the cellular mRNA 730 b and theoligonucleotides 725 b as templates. The extension products produced bythe reverse transcriptase can be subject to amplification andsequencing.

Sequencing reads can be subject to demultiplexing of cell labels,molecular labels, gene identities, and sample identities (e.g., in termsof sample indexing sequences of sample indexing oligonucleotides 725 aand 725 b). Demultiplexing of cell labels, molecular labels, and geneidentities can give rise to a digital representation of gene expressionof each single cell in the sample. Demultiplexing of cell labels,molecular labels, and sample identities, using sample indexing sequencesof sample indexing oligonucleotides, can be used to determine a sampleorigin.

In some embodiments, cellular component binding reagents againstcellular component binding reagents on the cell surface can beconjugated to a library of unique sample indexing oligonucleotides toallow cells to retain sample identity. For example, antibodies againstcell surface markers can be conjugated to a library of unique sampleindexing oligonucleotides to allow cells to retain sample identity. Thiswill enable multiple samples to be loaded onto the same Rhapsody™cartridge as information pertaining sample source is retained throughoutlibrary preparation and sequencing. Sample indexing can allow multiplesamples to be run together in a single experiment, simplifying andshortening experiment time, and eliminating batch effect.

Disclosed herein include methods for sample identification. In someembodiments, the method comprise: contacting one or more cells from eachof a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more cellular component targets, whereineach of the plurality of sample indexing compositions comprises acellular component binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; removingunbound sample indexing compositions of the plurality of sample indexingcompositions. The method can include barcoding (e.g., stochasticallybarcoding) the sample indexing oligonucleotides using a plurality ofbarcodes (e.g., stochastic barcodes) to create a plurality of barcodedsample indexing oligonucleotides; obtaining sequencing data of theplurality of barcoded sample indexing oligonucleotides; and identifyingsample origin of at least one cell of the one or more cells based on thesample indexing sequence of at least one barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides.

In some embodiments, the method for sample identification comprises:contacting one or more cells from each of a plurality of samples with asample indexing composition of a plurality of sample indexingcompositions, wherein each of the one or more cells comprises one ormore cellular component targets, wherein each of the plurality of sampleindexing compositions comprises a cellular component binding reagentassociated with a sample indexing oligonucleotide, wherein the cellularcomponent binding reagent is capable of specifically binding to at leastone of the one or more cellular component targets, wherein the sampleindexing oligonucleotide comprises a sample indexing sequence, andwherein sample indexing sequences of at least two sample indexingcompositions of the plurality of sample indexing compositions comprisedifferent sequences; removing unbound sample indexing compositions ofthe plurality of sample indexing compositions; and identifying sampleorigin of at least one cell of the one or more cells based on the sampleindexing sequence of at least one sample indexing oligonucleotide of theplurality of sample indexing compositions.

In some embodiments, identifying the sample origin of the at least onecell comprises: barcoding (e.g., stochastically barcoding) sampleindexing oligonucleotides of the plurality of sample indexingcompositions using a plurality of barcodes (e.g., stochastic barcodes)to create a plurality of barcoded sample indexing oligonucleotides;obtaining sequencing data of the plurality of barcoded sample indexingoligonucleotides; and identifying the sample origin of the cell based onthe sample indexing sequence of at least one barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides. In some embodiments, barcoding the sample indexingoligonucleotides using the plurality of barcodes to create the pluralityof barcoded sample indexing oligonucleotides comprises stochasticallybarcoding the sample indexing oligonucleotides using a plurality ofstochastic barcodes to create a plurality of stochastically barcodedsample indexing oligonucleotides.

In some embodiments, identifying the sample origin of the at least onecell can comprise identifying the presence or absence of the sampleindexing sequence of at least one sample indexing oligonucleotide of theplurality of sample indexing compositions. Identifying the presence orabsence of the sample indexing sequence can comprise: replicating the atleast one sample indexing oligonucleotide to generate a plurality ofreplicated sample indexing oligonucleotides; obtaining sequencing dataof the plurality of replicated sample indexing oligonucleotides; andidentifying the sample origin of the cell based on the sample indexingsequence of a replicated sample indexing oligonucleotide of theplurality of sample indexing oligonucleotides that correspond to theleast one barcoded sample indexing oligonucleotide in the sequencingdata.

In some embodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, ligating a replicating adaptorto the at least one barcoded sample indexing oligonucleotide.Replicating the at least one barcoded sample indexing oligonucleotidecan comprise replicating the at least one barcoded sample indexingoligonucleotide using the replicating adaptor ligated to the at leastone barcoded sample indexing oligonucleotide to generate the pluralityof replicated sample indexing oligonucleotides.

In some embodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, contacting a capture probewith the at least one sample indexing oligonucleotide to generate acapture probe hybridized to the sample indexing oligonucleotide; andextending the capture probe hybridized to the sample indexingoligonucleotide to generate a sample indexing oligonucleotide associatedwith the capture probe. Replicating the at least one sample indexingoligonucleotide can comprise replicating the sample indexingoligonucleotide associated with the capture probe to generate theplurality of replicated sample indexing oligonucleotides.

Daisy-Chaining Oligonucleotides

Disclosed herein include systems, methods, kits, and compositions forextending barcoded oligonucleotides (such as a sample indexingoligonucleotide, a control particle oligonucleotide, a controloligonucleotide, or an oligonucleotide conjugated to an antibody fordetermining protein expression), by PCR amplification of barcodedoligonucleotides using primers with long overhangs (e.g., 160 or morenucleotides in length). Such primers can be, for example, 200 or morenucleotides in length, such as Ultramer® DNA Oligonucleotides, Megamer®Single-Stranded DNA Fragments, or single-stranded DNA fragments ofgBlocks® Gene Fragments (Integrated DNA Technologies, Inc. (Coralville,Iowa)). The barcoded oligonucleotides can contain a common sequence(e.g., a common sequence that is 40 nucleotides in length) for the longprimer to bind and extend during PCR. In some embodiments, the longoverhang can include a secondary barcode sequence (e.g., a molecularlabel sequence) to add complexity to the sample. Thus, the length ofbarcoded oligonucleotides can be increased from, for example, 200-300nucleotides, to more than 400 nucleotides. Such longer barcodedoligonucleotides can survive ever increasing size selection during DNApurification using, for example, Solid Phase Reversible Immobilization(SPRI)_magnetic beads (Applied Biological Materials Inc. (Richmond,British Columbia, Canada)).

In some embodiments, oligonucleotides associated with (e.g., attachedto) cellular component binding reagents (e.g., antibody molecules) canbe barcoded (e.g., stochastically barcoded), after dissociation from thecellular component binding reagents, to generate the barcodedoligonucleotides. The barcoded oligonucleotides can be furtherlengthened using a primer with a long overhang. Thus, the lengths of theoligonucleotides associated with cellular component binding reagents andthe lengths of barcoded oligonucleotides, which may be limited by DNAsynthesis technologies or experimental design considerations, do notlimit the subsequent workflow (e.g., size selection during DNApurification). One non-limiting exemplary experimental designconsideration is the effects of long oligonucleotides attached tocellular component binding reagents on the reagents' binding specificityand/or efficacy. PCR daisy-chaining of barcoded oligonucleotides canovercome or address limitations of DNA synthesis technologies whilemaintaining antibody specificity and/or efficacy. The methods of thedisclosure for lengthening barcoded oligonucleotides with a commonsequence using a primer with a long overhang is referred to herein asdaisy-chaining the barcoded oligonucleotides using daisy-chainingamplification primers to generate daisy-chaining elongated amplicons.

Disclosed herein include methods for extending sample indexingoligonucleotides (or other barcoded oligonucleotides, such as controlparticle oligonucleotides). In some embodiments, after contacting one ormore cells from each of a plurality of samples with a sample indexingcomposition of a plurality of sample indexing compositions, the sampleindexing oligonucleotides can be barcoded using a plurality of barcodesto create a plurality of barcoded sample indexing oligonucleotides (SeeFIG. 7). The plurality of barcoded sample indexing oligonucleotides canbe amplified using a plurality of primers with long overhangs (referredto herein as daisy-chaining amplification primers) to create a pluralityof longer amplicons (referred to herein as daisy-chaining elongatedamplicons). Such longer amplicons can be, for example, substantiallylonger (e.g., 160 nucleotides longer) than the barcoded sample indexingoligonucleotides.

FIGS. 8A8 and 8A2 show a schematic illustration of an exemplary workflowof daisy-chaining a barcoded oligonucleotide. In some embodiments, asample indexing composition 805 can include a cellular component bindingreagent, such as an antibody, that is associated with a sample indexingoligonucleotide 825. The sample indexing oligonucleotide 825 can includea sample indexing sequence 825 s, a common region 825 d for binding to adaisy-chaining amplification primer (referred to herein as adaisy-chaining amplification primer-binding region or sequence 825 d),and a poly(A) tail 825 a. In some embodiments, the common region 825 dfor binding to a daisy-chaining amplification primer can be the same forall or some of sample indexing oligonucleotides 825. In someembodiments, the daisy-chaining amplification primer-binding region 825d of different sample indexing oligonucleotides 825 are different. Asample indexing oligonucleotide 825 can be an mRNA mimic. The sampleindexing sequences 825 s of at least two sample indexing compositions805 of the plurality of sample indexing compositions can comprisedifferent sequences.

The daisy-chaining amplification primer-binding region 825 d can bedifferent in different implementations, ranging from 10 to 200nucleotides in length. In some embodiments, daisy-chaining amplificationprimer-binding region 825 d can be, or be about, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, or a number or a range between any two ofthese values, nucleotides in length. In some embodiments, thedaisy-chaining amplification primer-binding region 825 d can be atleast, or be at most, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or200 nucleotides in length.

The sample indexing oligonucleotides 825 can be barcoded using aplurality of barcodes 815 (e.g., barcodes 815 associated with aparticle, such as a bead 81) to create a plurality of barcoded sampleindexing oligonucleotides 840. In some embodiments, a barcode 815 caninclude a poly(dT) region 815 t for binding to a sample indexingoligonucleotide 825, optionally a molecular label 815 m (e.g., fordetermining the number of occurrences of the sample indexingoligonucleotides), a cell label 815 c, and a universal label 815 u. Abarcoded sample indexing oligonucleotide 840 can include a reversecomplement 825 d_rt of the daisy-chaining amplification primer-bindingregion 825 d.

Barcoded sample indexing oligonucleotides 840 can be amplified usingforward and reverse primers 845, 850, including a plurality ofdaisy-chaining amplification primers 845, to create a plurality ofdaisy-chaining elongated amplicons 860. A daisy-chaining amplificationprimer 845 can comprise the sequence 845 d, a complement, a reversecomplement, or a combination thereof, of the daisy-chainingamplification primer-binding region 825 d (referred to herein asbarcoded sample indexing oligonucleotide-binding region 845 d). Adaisy-chaining amplification primer-binding region 825 d and a barcodedsample indexing oligonucleotide-binding region 845 d can be the samelengths, or different lengths. The barcoded sample indexingoligonucleotide-binding region 845 d of a daisy-chaining amplificationprimer 845 can bind to its reverse complementary sequence 825 d_rt on abarcoded sample indexing oligonucleotide 840. A daisy-chainingamplification primer 845 can include an overhang region 845 o (theoverhang region 845 o is an overhang of the daisy-chaining amplificationprimer 845 when the daisy-chaining amplification primer 845 binds to abarcoded sample indexing oligonucleotide 850). The plurality ofdaisy-chaining elongated amplicons 860, or a portion thereof, can besequenced for sample identification (or protein expression profiling,sequencing control, etc.). As illustrated in FIG. 8A1-8A2, a sampleindexing oligonucleotide 825 can be 200 nucleotides in length, abarcoded sample indexing oligonucleotides 840 can be 240 nucleotides inlength, and a daisy-chaining elongated amplicon 860 can be 400nucleotides in length.

The barcoded sample indexing oligonucleotide-binding region 845 d canhave different lengths in different implementations, for example,ranging from 10 to 200 nucleotides in length. In some embodiments, thebarcoded sample indexing oligonucleotide-binding region 845 d can be, orbe about, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or anumber or a range between any two of these values, nucleotides inlength. In some embodiments, the barcoded sample indexingoligonucleotide-binding region 845 d can be at least, or be at most, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides inlength.

In some embodiments, at least two daisy-chaining amplification primers845 can comprise barcoded sample indexing oligonucleotide-bindingregions 845 d with an identical sequence. Two or more daisy-chainingamplification primers 845 can comprise barcoded sample indexingoligonucleotide-binding regions 845 d with different sequences. Some orall of daisy-chaining amplification primers 845 can comprise barcodedsample indexing oligonucleotide-binding regions 845 d with an identicalsequence. The barcoded sample indexing oligonucleotide-binding regions845 d can comprise the daisy-chaining amplification primer bindingsequence 825 d, a complement thereof, a reverse complement thereof, or acombination thereof. Daisy-chaining amplification primer bindingsequences 825 d of at least two sample indexing compositions 805 cancomprise an identical sequence. Daisy-chaining amplification primerbinding sequences 825 d of at least two sample indexing compositions 805can comprise different sequences.

In some embodiments, the overhang region 845 o can comprise a barcodesequence (referred to herein as a daisy-chaining amplification primerbarcode sequence), such as a molecular label. For example, twodaisy-chaining amplification primers 845 o can comprise overhang regions845 o with an identical daisy-chaining amplification primer barcodesequence. As another example, two daisy-chaining amplification primers845 can comprise overhang regions 845 o with two differentdaisy-chaining amplification primer barcode sequences. Overhang regions845 o of daisy-chaining amplification primers 845 can comprise differentdaisy-chaining amplification primer barcode sequences.

The overhang region 845 can have different lengths in differentimplementations, for example, ranging from 50 to 1000 nucleotides inlength. In some embodiments, the overhang region 845 can be, or beabout, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or a numberor a range between any two of these values, nucleotides in length. Insome embodiments, the overhang region 845 can be at least, or be atmost, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000.

The daisy-chaining elongated amplicon 860 can have different lengths indifferent implementations, for example, ranging from 50 to 1000nucleotides in length. In some embodiments, the daisy-chaining elongatedamplicon 860 can be, or be about, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540,550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960,970, 980, 990, 1000 or a number or a range between any two of thesevalues, nucleotides in length. In some embodiments, the daisy-chainingelongated amplicon 860 can be at least, or be at most, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500,510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, or 1000.

FIGS. 8B1 and 8B2 show a schematic illustration of another exemplaryworkflow of daisy-chaining a barcoded oligonucleotide. In this workflow,the reverse complementary sequence of a switch oligonucleotide 860 canbe optionally added to a barcoded sample indexing oligonucleotide 840.During reverse transcription, upon reaching the 5′ end of the sampleindexing oligonucleotide 824, the terminal transferase activity of anenzyme (e.g., a reverse transcriptase, such as a Moloney murine leukemiavirus (MMLV)) adds a few additional nucleotides (mostly deoxycytidine)to the 3′ end of the newly synthesized strand 840. These bases canfunction as an anchoring site of the template switch (TS)oligonucleotide 860. Upon base pairing between the template switcholigonucleotide 860 and the appended deoxycytidine stretch, the enzyme“switches” template strands, from sample indexing oligonucleotide 825 tothe template switch oligonucleotide 860, and continues replication tothe 5′ end of the TS oligonucleotide 860. Thus, the resulting barcodedsample indexing oligonucleotide 840 contains a reverse complement of thesample indexing oligonucleotide 825 and the template switcholigonucleotide 860.

Barcoded sample indexing oligonucleotides 840 with a reverse complementsequence of the template switch oligonucleotide 860 can be amplifiedusing forward and reverse primers 845, 850 to create daisy-chainingelongated amplicons 860 with the reverse complement of the templateswitch oligonucleotide 860. One of the two primers can bind to the Read1 sequence 845 r 1, a universal sequence for amplification, which can beequivalent to the universal label 845 u in FIGS. 8A1-8A2. The secondprimer, a daisy-chaining amplification primers 845, can bind to thereverse complement sequence 825 d_rt of the daisy-chaining amplificationprimer-binding region 825 d. Accordingly, a long tail can be added tothe barcoded sample indexing oligonucleotide 860. After enzymaticfragmentation, end repair, A-tailing, and optionally sample index PCR,the resulting amplicons can include the P5 sequence 865 p 5, P7 sequence865 p 7, Read 1 sequence 815 r 1, and Read 2 sequence 865 r 2 for nextgeneration sequencing (e.g., using bridge amplification). As illustratedin FIGS. 8A1-8A2, a sample indexing oligonucleotide 825 can be 200nucleotides in length, a barcoded sample indexing oligonucleotides 840can be approximately 260 nucleotides in length, and a daisy-chainingelongated amplicon 860 can be more than 406 nucleotides in length.

Single Cell Sequencing Control Particles

Disclosed herein includes control particle compositions that can be usedfor, for example, single cell sequencing control. In some embodiments,the control particle composition comprises a plurality of controlparticle oligonucleotides associated with a control particle. Thecontrol particle associated with the plurality of control particleoligonucleotides is referred to herein also as a functionalized controlparticle. FIG. 9A is a non-limiting exemplary schematic illustration ofa particle functionalized with a plurality of oligonucleotides. FIG. 9Ashows that the control particle oligonucleotide associated with thecontrol particle can comprise a control barcode sequence and a poly(dA)region, mimicking a mRNA poly(A) tail. The control particleoligonucleotide can comprise a molecular label sequence, a binding sitefor a universal primer, or a combination thereof. The control particleoligonucleotides associated with the control particles can be the sameor different from one another. In some embodiments, at least two of thecontrol particle oligonucleotides associated with the control particlehave different control barcode sequence. In some embodiments, aplurality of a first control particle oligonucleotides and a pluralityof a second control oligonucleotides are associated with the controlparticle, wherein the first and the second particle oligonucleotideshave different control barcode sequence.

A bead, such as the CompBead™ Plus (BD (Franklin Lake, N.J.)) can befunctionalized with antibodies conjugated with oligonucleotides.CompBeads Plus are about 7.5 microns in size, which is similar to thesize of an immune cell. When functionalized with antibodies conjugatedwith oligonucleotides, CompBead Plus can be used as control cells orcontrol particles for single cell workflows. The AbO functionalized beadcan be used with any single cell workflow as a single cell sequencingcontrol.

Control Particle Oligonucleotide

The length of the control barcode sequence can be different in differentimplementations. In some embodiments, the control barcode sequence is,or is about, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990,1000, or a number or a range between any two of these values,nucleotides in length. In some embodiments, the control barcode sequenceis at least, or is at most, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128,130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540,550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960,970, 980, 990, or 1000 nucleotides in length.

The length of the control particle oligonucleotide can be different indifferent implementations. In some embodiments, the control particleoligonucleotide is, or is about, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500,510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000, or a number or a range betweenany two of these values, nucleotides in length. In some embodiments, thecontrol particle oligonucleotide is at least, or is at most, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730,740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870,880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000nucleotides in length.

In some embodiments, the number of the plurality of control particleoligonucleotides can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 1000000,10000000, 100000000, 1000000000, or a number or a range between any twoof these values. In some embodiments, the number of the plurality ofcontrol particle oligonucleotides can be at least, or be at most, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, 100000, 1000000, 10000000, 100000000, or 1000000000.

The plurality of control particle oligonucleotides can comprise the sameor different control barcode sequences. For example, at least two of theplurality of control particle oligonucleotides can comprise differentcontrol barcode sequences. In some embodiments, the control barcodesequences of at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 1000000,10000000, 100000000, or 1000000000 of the plurality of control particleoligonucleotides can be identical. In some embodiments, the controlbarcode sequences of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 1000000,10000000, 100000000, 1000000000, or a number or a range between any twoof these values, of the plurality of control particle oligonucleotidescan be identical.

The control barcode sequences of at least or at most 0.000000001%,0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%,0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100%, or a number or a range between any two of these values of theplurality of control particle oligonucleotides can be identical. Thecontrol barcode sequences of, or of about, 0.000000001%, 0.00000001%,0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a numberor a range between any two of these values, of the plurality of controlparticle oligonucleotides can be identical.

In some embodiments, the control barcode sequence is not homologous togenomic sequences of a species. The control barcode sequence can behomologous to genomic sequences of a species. The species can be anon-mammalian species. The non-mammalian species can be a phage species.The phage species can be T7 phage, a PhiX phage, or a combinationthereof.

Control Particle

In some embodiments, at least one of the plurality of control particleoligonucleotides is associated with the control particle through alinker. The at least one of the plurality of control particleoligonucleotides can comprise the linker. The linker can comprise achemical group. The chemical group can be reversibly or irreversiblyattached to the at least one of the plurality of control particleoligonucleotides. The chemical group can comprise a UV photocleavablegroup, a disulfide bond, a streptavidin, a biotin, an amine, a disulfidelinkage, or any combination thereof.

The diameter of the control particle can be, or be about, 1-1000micrometers, such as 10-100 micrometer or 7.5 micrometer. In someembodiments, the diameter of the control particle can be, or be about,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000 micrometers, or a number or arange between any two of these values. In some embodiments, the diameterof the control particle can be at least, or be at most, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 micrometers.

In some embodiments, the plurality of control particle oligonucleotidesis immobilized on the control particle. The plurality of controlparticle oligonucleotides can be partially immobilized on the controlparticle. The plurality of control particle oligonucleotides can beenclosed in the control particle. The plurality of control particleoligonucleotides can be partially enclosed in the control particle. Thecontrol particle can be disruptable.

In some embodiments, the control particle can be a bead. The bead canbe, or comprise, a Sepharose bead, a streptavidin bead, an agarose bead,a magnetic bead, a conjugated bead, a protein A conjugated bead, aprotein G conjugated bead, a protein A/G conjugated bead, a protein Lconjugated bead, an oligo(dT) conjugated bead, a silica bead, asilica-like bead, an anti-biotin microbead, an anti-fluorochromemicrobead, or any combination thereof. The control particle can comprisea material of polydimethylsiloxane (PDMS), polystyrene, glass,polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic,plastic, glass, methylstyrene, acrylic polymer, titanium, latex,Sepharose, cellulose, nylon, silicone, or any combination thereof. Thecontrol particle can comprise a disruptable hydrogel particle.

Protein Binding Reagent

In some embodiments, the control particle is associated with a pluralityof first protein binding reagents, and at least one of the plurality offirst protein binding reagents is associated with one of the pluralityof control particle oligonucleotides. FIG. 9B shows a non-limitingexemplary particle coated with a number of antibodies functionalizedwith oligonucleotides. The first protein binding reagent can comprise afirst antibody (e.g., a primary antibody, or a secondary antibody). Thecontrol particle oligonucleotide can be conjugated to the first proteinbinding reagent through a linker. The first control particleoligonucleotide can comprise the linker. The linker can comprise achemical group. The chemical group can be reversibly or irreversiblyattached to the first protein binding reagent. The chemical group cancomprise a UV photocleavable group, a disulfide bond, a streptavidin, abiotin, an amine, a disulfide linkage, or any combination thereof.

In some embodiments, the control particle is associated with a pluralityof second protein binding reagents. At least one of the plurality ofsecond protein binding reagents can be associated with one of theplurality of control particle oligonucleotides. FIG. 9C shows anon-limiting exemplary particle coated with a plurality of firstantibodies functionalized with oligonucleotides and a plurality ofsecond antibodies not functionalized with oligonucleotides. Theantibodies on the control particle can be titrated with ratios of hotantibodies (e.g., associated with control particle oligonucleotide) andcold antibodies (e.g., not associated with control particleoligonucleotides) to alter the amount of sequencing reads obtained froma control particle. The first antibodies and the second antibodies canbe the same or different.

FIG. 9D is a non-limiting exemplary schematic illustration of a particlefunctionalized with a plurality of first control particleoligonucleotides, a plurality of second control particleoligonucleotides conjugated to a plurality of second antibodies and aplurality of third control particle oligonucleotides with relative high,medium, and low abundance. The plurality of first control particleoligonucleotides can be conjugated to a plurality of first proteinbinding reagents. The plurality of second control particleoligonucleotides can be conjugated to a plurality of second proteinbinding reagents. The plurality of third control particleoligonucleotides can be conjugated to a plurality of third proteinbinding reagents.

The relative abundance of the first, second, and third control particleoligonucleotides can mimic mRNAs with different expression levels. Thecontrol particle oligonucleotide associated with the first proteinbinding reagent and the control particle oligonucleotide associated withthe second protein binding reagent can comprise different controlbarcode sequences. Different protein binding reagents, such asantibodies, and the different control particle oligonucleotides on thecontrol particle can be titrated to generate a standard curve. The firstprotein binding reagents, the second protein binding reagents, or thethird protein binding reagents can be identical or different proteinbinding reagents.

In some embodiments, the ratio of the number of the plurality of firstcontrol particle oligonucleotides and the number of the plurality ofsecond (or third) control particle oligonucleotides can be, or be about,1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2,1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26,1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38,1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50,1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62,1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74,1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86,1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98,1:99, 1:100, or a number or a range between any two of these numbers. Insome embodiments, the ratio of the number of the plurality of firstcontrol particle oligonucleotides and the number of the plurality ofsecond (or third) control particle oligonucleotides can be at least, orbe at most, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8,1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12,1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24,1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36,1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48,1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60,1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72,1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84,1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96,1:97, 1:98, 1:99, or 1:100.

In some embodiments, the first protein binding reagent can be associatedwith a partner binding reagent (e.g., a secondary antibody), and thefirst protein binding reagent is associated with the control particleusing the partner binding reagent. The partner binding reagent cancomprise a partner antibody. The partner antibody can comprise ananti-cat antibody, an anti-chicken antibody, an anti-cow antibody, ananti-dog antibody, an anti-donkey antibody, an anti-goat antibody, ananti-guinea pig antibody, an anti-hamster antibody, an anti-horseantibody, an anti-human antibody, an anti-llama antibody, an anti-monkeyantibody, an anti-mouse antibody, an anti-pig antibody, an anti-rabbitantibody, an anti-rat antibody, an anti-sheep antibody, or a combinationthereof. The partner antibody can comprise an immunoglobulin G (IgG), aF(ab′) fragment, a F(ab′)2 fragment, a combination thereof, or afragment thereof.

In some embodiments, the first protein binding reagent is associatedwith two or more of the plurality of control particle oligonucleotideswith an identical control barcode sequence. The first protein bindingreagent can be associated with two or more of the plurality of controlparticle oligonucleotides with different control barcode sequences. Insome embodiments, at least one of the plurality of first protein bindingreagents is not associated with any of the plurality of control particleoligonucleotides. The first protein binding reagent associated with thecontrol particle oligonucleotide and the first protein binding reagentnot associated with any control particle oligonucleotide can beidentical protein binding reagents.

Control Barcode Diversity

The plurality of control particle oligonucleotides associated with onecontrol particle can comprise a number of control particleoligonucleotides with different control barcode sequences. The number ofcontrol barcode sequences that control particle oligonucleotides havecan be different in different implementation. In some embodiments, thenumber of control barcode sequences that the control particleoligonucleotides have can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 10000, 100000, 1000000, or a number or a range betweenany two of these values. In some embodiments, the number of controlbarcode sequences that the control particle oligonucleotides have can beat least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,10000, 100000, or 1000000.

In some embodiments, the number of control particle oligonucleotideswith the same control particle oligonucleotide sequence can be, or beabout, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 100000,1000000, or a number or a range between any two of these values. In someembodiments, the number of control particle oligonucleotides with thesame control particle oligonucleotide sequence can be at least, or be atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 100000, or1000000.

In some embodiments, the plurality of control particle oligonucleotidescomprises a plurality of first control particle oligonucleotides eachcomprising a first control barcode sequence, and a plurality of secondcontrol particle oligonucleotides each comprising a second controlbarcode sequence, and the first control barcode sequence and the secondcontrol barcode sequence have different sequences. The number of theplurality of first control particle oligonucleotides and the number ofthe plurality of second control particle oligonucleotides can be aboutthe same. The number of the plurality of first control particleoligonucleotides and the number of the plurality of second controlparticle oligonucleotides can be different. The number of the pluralityof first control particle oligonucleotides can be at least 2 times, 10times, 100 times, or more greater than the number of the plurality ofsecond control particle oligonucleotides. In some embodiments, the ratioof the number of the plurality of first control particleoligonucleotides and the number of the plurality of second controlparticle oligonucleotides can be, or be about, 1:1, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29,1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41,1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53,1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65,1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77,1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89,1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000,1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or anumber or a range between any two of the values. In some embodiments,the ratio of the number of the plurality of first control particleoligonucleotides and the number of the plurality of second controlparticle oligonucleotides can be at least, or be at most, 1:1, 1:1.1,1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16,1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28,1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40,1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52,1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64,1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76,1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88,1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000,1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, or 1:10000.

Detectable Moiety

In some embodiments, the control particle is associated with adetectable moiety, for example an optical moiety, such as a fluorophoreor a chromophore. The control particle oligonucleotide can be associatedwith a detectable moiety, for example an optical moiety. In someembodiments, the first protein binding reagent can be associated with anoptical moiety (FIG. 9E). The second protein binding reagent can beassociated with an optical moiety. A control particle associated with anoptical moiety (e.g., a bead fluorescently tagged) can also be used forimaging and flow cytometry.

The detectable moiety can be selected from a group ofspectrally-distinct detectable moieties. Spectrally-distinct detectablemoieties include detectable moieties with distinguishable emissionspectra even if their emission spectral may overlap. Non-limitingexamples of detectable moieties include Xanthene derivatives:fluorescein, rhodamine, Oregon green, eosin, and Texas red; Cyaninederivatives: cyanine, indocarbocyanine, oxacarbocyanine,thiacarbocyanine, and merocyanine; Squaraine derivatives andring-substituted squaraines, including Seta, SeTau, and Square dyes;Naphthalene derivatives (dansyl and prodan derivatives); Coumarinderivatives; oxadiazole derivatives: pyridyloxazole, nitrobenzoxadiazoleand benzoxadiazole; Anthracene derivatives: anthraquinones, includingDRAQ5, DRAQ7 and CyTRAK Orange; Pyrene derivatives: cascade blue;Oxazine derivatives: Nile red, Nile blue, cresyl violet, oxazine 170;Acridine derivatives: proflavin, acridine orange, acridine yellow;Arylmethine derivatives: auramine, crystal violet, malachite green; andTetrapyrrole derivatives: porphin, phthalocyanine, bilirubin. Othernon-limiting examples of detectable moieties include Hydroxycoumarin,Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, PacificOrange, Lucifer yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates,PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, Fluorescein,BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine,Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), APC-Cy7conjugates, Hoechst 33342, DAPI, Hoechst 33258, SYTOX Blue, ChromomycinA3, Mithramycin, YOYO-1, Ethidium Bromide, Acridine Orange, SYTOX Green,TOTO-1, TO-PRO-1, TO-PRO: Cyanine Monomer, Thiazole Orange, CyTRAKOrange, Propidium Iodide (PI), LDS 751, 7-AAD, SYTOX Orange, TOTO-3,TO-PRO-3, DRAQ5, DRAQ7, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, and SNARF.

The excitation wavelength of the detectable moieties can vary, forexample be, or be about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530,540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670,680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810,820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,960, 970, 980, 990, 1000 nanometers, or a number or a range between anytwo of these values. The emission wavelength of the detectable moietiescan also vary, for example be, or be about, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500,510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000 nanometers, or a number or arange between any two of these values.

The molecular weights of the detectable moieties can vary, for examplebe, or be about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, 1000 Daltons (Da), or a number or a range between any two ofthese values. The molecular weights of the detectable moieties can alsovary, for example be, or be about, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790,800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930,940, 950, 960, 970, 980, 990, 1000 kilo Daltons (kDa), or a number or arange between any two of these values.

The group of spectrally distinct detectable moieties can, for example,include five different fluorophores, five different chromophores, acombination of five fluorophores and chromophores, a combination of fourdifferent fluorophores and a non-fluorophore, a combination of fourchromophores and a non-chromophore, or a combination of fourfluorophores and chromophores and a non-fluorophore non-chromophore. Insome embodiments, the detectable moieties can be one of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10000, or a number or a rangebetween any two of these values, of spectrally-distinct moieties.

Control Particle Workflow

The AbO functionalized bead can be used with any single cell workflow asa single cell sequencing control. Single cell workflows can utilizemicrowell arrays or microwell cartridges (e.g., BD Rhapsody™) ormicrofluidics devices (e.g., 10× Genomics (San Francisco, Calif.),Drop-seq (McCarroll Lab, Harvard Medical School (Cambridge,Massachusett); Macosko et al., Cell, 2015 May 21 16; 5:1202, the contentof which is incorporated herein by reference in its entirety), or Abseq(Mission Bio (San Francisco, Calif.); Shahi et al., Sci Rep. 2017 Mar.14; 7:44447, the content of which is hereby incorporated by reference inits entirety) in combination with solid or semisolid particlesassociated with stochastic barcodes (e.g., BD Rhapsody, or Drop-seq) ordisruptable hydrogel particles enclosing releasable stochastic barcodes(e.g., 10× Genomics, or Abseq). The functionalized bead can be a controlfor determining efficiency of single cell workflows, analogous toexternal RNA control consortiums (ERCCs) being used for bulk RNAseq ormicroarray studies.

Disclosed herein include methods for determining the numbers of targetsusing a plurality of control particle oligonucleotides. The methods fordetermining the number of targets (e.g., gene expression) can be usedwith other methods disclosed herein. For example, a workflow can be usedfor determining protein expression and gene expression using a pluralityof control particle oligonucleotides. In some embodiments, the methodcomprises: barcoding (e.g., stochastically barcoding) a plurality oftargets of a cell of a plurality of cells and a plurality of controlparticle oligonucleotides using a plurality of barcodes (e.g.,stochastic barcodes) to create a plurality of barcoded targets (e.g.,stochastically barcoded targets) and a plurality of barcoded controlparticle oligonucleotides (e.g., stochastically barcoded controlparticle oligonucleotides), wherein each of the plurality of barcodescomprises a cell label sequence, a barcode sequence (e.g., a molecularlabel), and a target-binding region, wherein at least two barcodes ofthe plurality of barcodes comprise different barcode sequences (e.g.,molecular labels), and wherein at least two barcodes of the plurality ofbarcodes comprise an identical cell label sequence, wherein a controlparticle composition comprises a control particle associated with theplurality of control particle oligonucleotides, wherein each of theplurality of control particle oligonucleotides comprises a controlbarcode sequence and a pseudo-target region comprising a sequencesubstantially complementary to the target-binding region of at least oneof the plurality of barcodes. The method can comprise: obtainingsequencing data of the plurality of barcoded targets and the pluralityof barcoded control particle oligonucleotides; counting the number ofbarcode sequences (e.g., molecular labels) with distinct sequencesassociated with the plurality of control particle oligonucleotides withthe control barcode sequence in the sequencing data. The method cancomprise: for at least one target of the plurality of targets: countingthe number of barcode sequences with distinct sequences associated withthe target in the sequencing data; and estimating the number of thetarget, wherein the number of the target estimated correlates with thenumber of barcodes sequences (e.g., molecular labels) with distinctsequences associated with the target counted and the number of barcodesequences (e.g., molecular labels) with distinct sequences associatedwith the control barcode sequence. In some embodiments, thepseudo-target region comprises a poly(dA) region. The pseudo-targetregion can comprise a subsequence of the target.

In some embodiments, the number of the target estimated can correlatewith the number of barcode sequences (e.g., molecular labels) withdistinct sequences associated with the target counted, the number ofbarcode sequences (e.g., molecular labels) with distinct sequencesassociated with the control barcode sequence, and the number of theplurality of control particle oligonucleotides comprising the controlbarcode sequence. The number of the target estimated can correlate withthe number of barcode sequences (e.g., molecular labels) with distinctsequences associated with the target counted, and a ratio of the numberof the plurality of control particle oligonucleotides comprising thecontrol barcode sequence and the number of barcode sequences (e.g.,molecular labels) with distinct sequences associated with the controlbarcode sequence.

For example, if the control particle has 100 control particleoligonucleotides with a particular control barcode sequence and thenumber of barcode sequences (e.g., molecular labels) with distinctsequences associated with the control barcode sequence (e.g., the numberof control particle oligonucleotides with the control barcode sequencethat survive the library preparation process) is 80, then the efficiencyof the library preparation (e.g., reverse transcription, amplification,etc.) is 80%. Thus, data from different library preparations can becompared by normalizing using the library preparation efficiency.

As another example, the control particle can comprise five controlparticle oligonucleotides with a particular control barcode sequencingmimicking a low expression gene. If the number of barcode sequences(e.g., molecular labels) with distinct sequences associated with thecontrol barcode sequence is five, and a low expression gene is notdetected, then a conclusion that the low expression gene is notexpressed (or the cell has fewer than five mRNAs of the gene) can bemade. However, if the number of barcode sequences (e.g., molecularlabels) with distinct sequences associated with the control barcodesequence is zero, and a low expression gene is not detected, then aconclusion that the low expression gene is not expressed cannot be made.

Capture efficiency can be determined for control particleoligonucleotides with different abundance. Normalization can beperformed based on the capture efficiency of control particleoligonucleotides with two or more control barcode sequences. In someembodiments, counting the number of barcode sequences (e.g., molecularlabels) with distinct sequences associated with the plurality of controlparticle oligonucleotides with the control barcode sequence in thesequencing data comprises: counting the number of barcode sequences(e.g., molecular labels) with distinct sequences associated with thefirst control barcode sequence in the sequencing data; and counting thenumber of barcode sequences (e.g., molecular labels) with distinctsequences associated with the second control barcode sequence in thesequencing data. The number of the target estimated can correlate withthe number of barcode sequences (e.g., molecular labels) with distinctsequences associated with the target counted, the number of barcodesequences (e.g., molecular labels) with distinct sequences associatedwith the first control barcode sequence, and the number of barcodesequences (e.g., molecular labels) with distinct sequences associatedwith the second control barcode sequence.

In some embodiments, the method comprises releasing the at least one ofthe plurality of control particle oligonucleotides from the controlparticle prior to stochastically barcoding the plurality of targets andthe control particle and the plurality of control particleoligonucleotides.

In some embodiments, barcoding (e.g., stochastically barcoding) theplurality of targets and the plurality of control particleoligonucleotides using the plurality of barcodes (e.g., stochasticbarcodes) comprises: contacting the plurality of barcodes with targetsof the plurality of targets and control particle oligonucleotides of theplurality of control particle oligonucleotides to generate barcodeshybridized to the targets and the control particle oligonucleotides; andextending the barcodes hybridized to the targets and the controlparticle oligonucleotides to generate the plurality of barcoded targetsand the plurality of barcoded control particle oligonucleotides (e.g.,the plurality of stochastically barcoded targets and the plurality ofstochastically barcoded control particle oligonucleotides). Extendingthe barcodes can comprise extending the barcodes using a DNA polymerase,a reverse transcriptase, or a combination thereof.

In some embodiments, the method comprises amplifying the plurality ofstochastically barcoded targets and the plurality of stochasticallybarcoded control particle oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of stochastically barcoded targetsand the plurality of stochastically barcoded control particleoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of the barcode sequence (e.g.,molecular label) and at least a portion of the control particleoligonucleotide or at least a portion of the barcode sequence (e.g.,molecular label) and at least a portion of the control particleoligonucleotide. Obtaining the sequencing data can comprise obtainingsequencing data of the plurality of amplicons. Obtaining the sequencingdata can comprise sequencing the at least a portion of the barcodesequence (e.g., molecular label) and the at least a portion of thecontrol particle oligonucleotide, or the at least a portion of thebarcode sequence (e.g., molecular label) and the at least a portion ofthe control particle oligonucleotide.

Microwell Cartridge or Array Workflow

FIG. 10 is a schematic illustration of an exemplary workflow of usingparticles functionalized with oligonucleotides for single cellsequencing control. In some embodiments, a control particle compositioncomprises a plurality of control particle oligonucleotides associatedwith a control particle 1004. For example, a control particle 1040 canbe associated with a control particle oligonucleotide 1025 conjugated toan antibody 1005 bound to the control particle 1040. A plurality ofcontrol particles 1040 functionalized with control particleoligonucleotides 1025 can be spiked into a plurality of cells at, forexample, 5%. Control particles 1040 can be treated as “cells” in thesubsequent workflow. The control particles 1040 can also be referred toas control cells or control cell particles. Cells 1010 and the controlparticles 1040 can be then separated into a plurality of compartments,such as wells of a microwell array, wherein a single compartment 1015 a,1015 b is sized to fit a single cell or control particle and a singlebead 1020 a, 1020 b. Beads 1020 a, 1020 b can be loaded into thecompartments 1015 a, 1015 b

Each bead can comprise a plurality of oligonucleotide probes, which cancomprise a cell label that is common to all oligonucleotide probes on abead, and barcode sequences (e.g., molecular label sequences). In someembodiments, each oligonucleotide probe can comprise a target bindingregion, for example, a poly(dT) sequence. The oligonucleotides 1025conjugated to the antibody 1005 can be detached from the antibody 1005using chemical, optical or other means. The cell 1010 can be lysed 1035to release nucleic acids within the cell, such as genomic DNA orcellular mRNA 1030. Cellular mRNAs 1030 and control particleoligonucleotides 1025 can be captured by the oligonucleotide probes onbeads 1020 a, 1020 b respectively, for example, by hybridizing to thepoly(dT) sequence. Beads can be retrieved and the captured cellularmRNAs 1030 and control particle oligonucleotides 1025 (e.g.,corresponding to around 5000 cells in total) can be pooled.

A reverse transcriptase can be used to extend the oligonucleotide probeshybridized to the cellular mRNA 1030 and the control particleoligonucleotides 1025 using the cellular mRNA 1030 and theoligonucleotides 1025 as templates. The extension products produced bythe reverse transcriptase can be subject to amplification andsequencing. Sequencing reads can be subject to demultiplexing of a celllabel, a barcode sequence (e.g., a molecular label), gene identity,control particle oligonucleotide sequence, etc., which can be used todetermine single cell gene expression profiles and quantity efficiencyof the entire or part of the workflow (e.g., cell capture efficiency).For example, the number of control particles captured can be determinedbased on the number of cell labels associated with the control barcodesequence in the data. The efficiency of the workflow can be a ratio ofthe number of control particles captured and the number of controlparticles spiked in.

Microfluidics Workflow

FIG. 10 is a schematic illustration of another exemplary workflow ofusing particles functionalized with oligonucleotides for single cellsequencing control. A plurality of control particles 1040 functionalizedwith control particle oligonucleotides 1025 can be spiked into aplurality of cells at, for example, 5%. Control particles 1040 can betreated as “cells” in the subsequent workflow. The control particles1040 can also be referred to as control cells or control cell particles.Cells 1010 and the control particles 1040 can be then separated using amicrofluidics device into a plurality of compartments, such as droplets1045 a, 1045. Each droplet 1045 a, 1045 b can include one cell 1010 orone control particle 1040 and a hydrogel bead 1020 a, 1020 b.

Each bead 1020 a, 1020 b can comprise a plurality of oligonucleotideprobes, which can comprise a cell label that is common to alloligonucleotide probes on a bead, and barcode sequences (e.g., molecularlabel sequences). In some embodiments, each oligonucleotide probe cancomprise a target binding region, for example, a poly(dT) sequence. Thebead 1020 a, 1020 b can include reagents for the subsequent workflow(e.g., reverse transcription). The oligonucleotides 1025 conjugated tothe antibody 1005 can be detached from the antibody 1005 using chemical,optical or other means. The cell 1010 can be lysed 1035 to releasenucleic acids within the cell, such as genomic DNA or cellular mRNA1030. Cellular mRNAs 630 and control particle oligonucleotides 1025 canbe captured by the oligonucleotide probes released from beads 1020 a,1020 b respectively, for example, by hybridizing to the poly(dT)sequence. A reverse transcriptase can be used to extend theoligonucleotide probes hybridized to the cellular mRNA 1030 and theoligonucleotides 1025 using the cellular mRNA 1030 and theoligonucleotides 1025 as templates.

After breaking up the droplets 1045 a, 1045 b, the extension productsproduced by the reverse transcriptase can be pooled and subject toamplification and sequencing. Sequencing reads can be subject todemultiplexing of cell label, molecular label, gene identity, controlparticle oligonucleotide sequence, etc. to determine single cell geneexpression profiles and quantity efficiency of the entire or part of theworkflow.

Control Oligonucleotides for Determining Single Cell SequencingEfficiency

Also disclosed herein include methods, kits and systems for determiningsingle cell sequencing efficiency. Such methods, kits and systems can beused in, or in combination with, any suitable methods, kits and systemsdisclosed herein, such as the methods, kits and systems for measuringcellular component expression level (for example protein expressionlevel) using cellular component binding reagents associated witholigonucleotides.

In some embodiments, by labeling single cells with antibodies conjugatedwith oligonucleotides (e.g., with a universal antibody or biomarkerantibody) and generating next generation sequencing libraries with them,the signals from the oligonucleotides in NGS reads can be used todetermine single cell NGS efficiency. This can then be used as a QC stepor an evaluation tool for efficacy for different single cell sequencingplatforms. For example, the control oligonucleotides can be used in anysuitable methods, kits and systems disclosed herein, for example themethods, kits and systems for measuring cellular component expressionlevel (for example protein expression level) using cellular componentbinding reagents associated with oligonucleotides.

Antibodies conjugated with oligonucleotides (referred to herein as“AbOs” can be used with any single cell workflow as a single cellsequencing control. Single cell workflows can utilize microwell arraysor microwell cartridges (e.g., BD Rhapsody™) or microfluidics devices(e.g., 10× Genomics (San Francisco, Calif.), Drop-seq (McCarroll Lab,Harvard Medical School (Cambridge, Mass.); Macosko et al., Cell, 2015May 21 16; 5:1202, the content of which is incorporated herein byreference in its entirety), or Abseq (Mission Bio (San Francisco,Calif.); Shahi et al., Sci Rep. 2017 Mar. 14; 7:44447, the content ofwhich is hereby incorporated by reference in its entirety) incombination with solid or semisolid particles associated with barcodes,such as stochastic barcodes (e.g., BD Rhapsody, or Drop-seq), ordisruptable hydrogel particles enclosing releasable barcodes, such asstochastic barcodes (e.g., 10× Genomics, or Abseq). AbOs can be acontrol for determining efficiency of single cell workflows. Forexample, the single cell sequencing platform from 10× Genomics performssingle cell capture using emulsions to encapsulate single cells indroplets. Because these droplets cannot be easily visualized, captureefficiency of single cells cannot be easily determined. The use of AbOsupstream of such single cell sequencing workflow allows users toevaluate the single cell capture efficiency after sequencing and rate ofdoublet formation.

FIG. 11 shows a schematic illustration of an exemplary workflow of usingcontrol oligonucleotide-conjugated antibodies for determining singlecell sequencing efficiency. In some embodiments, one or more cells(e.g., 5000) 1100 can be stained with an antibody 1105 conjugated with acontrol oligonucleotide 1125 prior to being loading onto a microwell1115 of a microwell cartridge or array. Cells 1110 can be then separatedinto a plurality of compartments, such as wells of a microwell array,wherein a single compartment 1115 is sized to fit a single cell and asingle bead 1120.

The bead can, for example, comprise a plurality of oligonucleotideprobes, which can comprise a cell label that is common to alloligonucleotide probes on a bead, and barcode sequences (e.g., molecularlabel sequences). In some embodiments, each oligonucleotide probe cancomprise a target binding region, for example, a poly(dT) sequence. Theoligonucleotides 1125 conjugated to the antibody 1105 can be detachedfrom the antibody 1105 using chemical, optical or other means. The cell1110 can be lysed 1135 to release nucleic acids within the cell, such asgenomic DNA or cellular mRNA 1130. Cellular mRNAs 1130 and controloligonucleotides 1125 can be captured by the oligonucleotide probes on abead 1120, for example, by hybridizing to the poly(dT) sequence. Beadscan be retrieved and the captured cellular mRNAs 1130 (e.g.,corresponding to around 5000 cells in total) can be pooled.

A reverse transcriptase can be used to extend the oligonucleotide probeshybridized to the cellular mRNA 1130 and the oligonucleotides 1125 usingthe cellular mRNA 1130 and the oligonucleotides 1125 as templates. Theextension products produced by the reverse transcriptase can be subjectto amplification and sequencing. Sequencing reads can be subject todemultiplexing of a cell label, a barcode sequence (e.g., a molecularlabel), gene identity, control oligonucleotide sequence, etc. todetermine single cell gene expression profiles and quantity efficiencyof the entire or part of the workflow (e.g., cell capture efficiency).The number of cells that are captured and go through the librarypreparation successfully (e.g., fewer than 5000 cells) can bedetermined.

FIG. 12 shows another schematic illustration of an exemplary workflow ofusing control oligonucleotide-conjugated antibodies for determiningsingle cell sequencing efficiency. In FIG. 12, droplets 1245 a, 1245 bcontaining a single cell 1210 a, 1210 b and a single particle 1220 a,1220 b can be formed using a microfluidic device. The single cells 1210a, 1210 b can be bound to antibodies 1205 a, 1205 b conjugated withcontrol oligonucleotides 1225 a, 1225 b. After cell lysis and reversetranscription in droplets 1245 a, 1245 b, droplets can be broken up andthe content pooled for library preparation. The number of cells that arecaptured and go through the library preparation successfully can bedetermined.

FIGS. 13A-13C are plots showing that control oligonucleotides can beused for cell counting. FIGS. 13A-13B show that control oligonucleotidesof AbOs can be used as a control for cell counting. The falling pointsof the mRNA counts plot and the control oligonucleotide counts plot cancoincide if 100% capture and library preparation efficiency is achieved.FIG. 13C shows that using conventional mRNA-only cell label calling maymiss transcriptionally low cells in the mist of transcriptionally highcells. This method may call cutoff at n cell labels. This may occur whenquiescent T cells within a large population of activated T cells, whereactivated T cells can have several fold higher in RNA transcription.This may also occur when in a targeted panel (e.g., cancer panel),non-targeted cells (non-cancer cells) with low expression of targetedgenes are going to be dropped off. However, since protein expression ismuch higher, transcriptionally low cells still have higher chance to becalled. This method may call cutoff at m cell labels, where m>n.

Disclosed herein include methods for sequencing control. For example,the methods can be used for determining single cell sequencingefficiency. The methods for determining single cell sequencingefficiency can be used with other methods disclosed herein. For example,the method for used for single cell sequencing efficiency can be usedwith the method for determining protein expression. As another example,a workflow can be used for determining single cell sequencingefficiency, protein expression, and/or gene expression.

In some embodiments, the method comprises: contacting one or more cellsof a plurality of cells with a control composition of a plurality ofcontrol compositions, wherein a cell of the plurality of cells comprisesa plurality of targets and a plurality of protein targets, wherein eachof the plurality of control compositions comprises a protein bindingreagent associated with a control oligonucleotide, wherein the proteinbinding reagent is capable of specifically binding to at least one ofthe plurality of protein targets, and wherein the controloligonucleotide comprises a control barcode sequence and a pseudo-targetregion comprising a sequence substantially complementary to thetarget-binding region of at least one of the plurality of barcodes;barcoding the control oligonucleotides using a plurality of barcodes tocreate a plurality of barcoded control oligonucleotides, wherein each ofthe plurality of barcodes comprises a cell label sequence, a barcodesequence (e.g., a molecular label sequence), and a target-bindingregion, wherein the barcode sequences (e.g., the molecular labelsequences) of at least two barcodes of the plurality of barcodescomprise different sequences, and wherein at least two barcodes of theplurality of barcodes comprise an identical cell label sequence;obtaining sequencing data of the plurality of barcoded controloligonucleotides; determining at least one characteristic (e.g., thenumber of cells that are captured and go through the library preparationsuccessfully) of the one or more cells using at least one characteristicof the plurality of barcoded control oligonucleotides in the sequencingdata. In some embodiments, the pseudo-target region comprises a poly(dA)region.

In some embodiments, determining the at least one characteristic of theone or more cells comprises: determining the number of cell labelsequences with distinct sequences associated with the plurality ofbarcoded control oligonucleotides in the sequencing data; anddetermining the number of the one or more cells using the number of celllabel sequences with distinct sequences associated with the plurality ofbarcoded control oligonucleotides. The method can comprise: determiningsingle cell capture efficiency based the number of the one or more cellsdetermined. The method can comprise: comprising determining single cellcapture efficiency based on the ratio of the number of the one or morecells determined and the number of the plurality of cells.

In some embodiments, determining the at least one characteristic of theone or more cells using the characteristics of the plurality of barcodedcontrol oligonucleotides in the sequencing data comprises: for each celllabel in the sequencing data, determining the number of barcodesequences (e.g., molecular label sequences) with distinct sequencesassociated with the cell label and the control barcode sequence; anddetermining the number of the one or more cells using the number ofbarcode sequences with distinct sequences associated with the cell labeland the control barcode sequence. Determining the number of barcodesequences with distinct sequences associated with the cell label and thecontrol barcode sequence can comprise: for each cell label in thesequencing data, determining the number of barcode sequences with thehighest number of distinct sequences associated with the cell label andthe control barcode sequence. Determining the number of the one or morecells using the number of barcode sequences with distinct sequencesassociated with the cell label and the control barcode sequence cancomprise: generating a plot of the number of barcode sequences with thehighest number of distinct sequences with the number of cell labels inthe sequencing data associated with the number of barcode sequences withthe highest number of distinct sequences; and determining a cutoff inthe plot as the number of the one or more cells.

In some embodiments, the method comprises releasing the controloligonucleotide from the cellular component binding reagent (e.g., theprotein binding reagent) prior to barcoding the controloligonucleotides. In some embodiments, the method comprises removingunbound control compositions of the plurality of control compositions.Removing the unbound control compositions can comprise washing the oneor more cells of the plurality of cells with a washing buffer. Removingthe unbound unbound control compositions can comprise selecting cellsbound to at least one cellular component binding reagent of the controlcomposition using flow cytometry.

In some embodiments, barcoding the control oligonucleotides comprises:barcoding the control oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded controloligonucleotides. In some embodiments, barcoding the plurality ofcontrol oligonucleotides using the plurality of barcodes comprises:contacting the plurality of barcodes with control oligonucleotides ofthe plurality of control compositions to generate barcodes hybridized tothe control oligonucleotides; and extending the stochastic barcodeshybridized to the control oligonucleotides to generate the plurality ofbarcoded control oligonucleotides. Extending the barcodes can compriseextending the barcodes using a DNA polymerase, a reverse transcriptase,or a combination thereof. In some embodiments, the method comprisesamplifying the plurality of barcoded control oligonucleotides to producea plurality of amplicons. Amplifying the plurality of barcoded controloligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of the stochastic barcode sequence(e.g., a molecular label sequence) and at least a portion of the controloligonucleotide. In some embodiments, obtaining the sequencing datacomprises obtaining sequencing data of the plurality of amplicons.Obtaining the sequencing data can comprise sequencing the at least aportion of the molecular label sequence and the at least a portion ofthe control oligonucleotide.

Cell Overloading and Multiplet Identification

Also disclosed herein include methods, kits and systems for identifyingcell overloading and multiplet. Such methods, kits and systems can beused in, or in combination with, any suitable methods, kits and systemsdisclosed herein, for example the methods, kits and systems formeasuring cellular component expression level (such as proteinexpression level) using cellular component binding reagents associatedwith oligonucleotides.

Using current cell-loading technology, when about 20000 cells are loadedinto a microwell cartridge or array with ˜60000 microwells, the numberof microwells or droplets with two or more cells (referred to asdoublets or multiplets) can be minimal. However, when the number ofcells loaded increases, the number of microwells or droplets withmultiple cells can increase significantly. For example, when about 50000cells are loaded into about 60000 microwells of a microwell cartridge orarray, the percentage of microwells with multiple cells can be quitehigh, such as 11-14%. Such loading of high number of cells intomicrowells can be referred to as cell overloading. However, if the cellsare divided into a number of groups (e.g., 5), and cells in each groupare labeled with sample indexing oligonucleotides with distinct sampleindexing sequences, a cell label (e.g., a cell label of a barcode, suchas a stochastic barcode) associated with two or more sample indexingsequences can be identified in sequencing data and removed fromsubsequent processing. In some embodiments, the cells are divided into alarge number of groups (e.g., 10000), and cells in each group arelabeled with sample indexing oligonucleotides with distinct sampleindexing sequences, a sample label associated with two or more sampleindexing sequences can be identified in sequencing data and removed fromsubsequent processing. In some embodiments, different cells are labeledwith cell identification oligonucleotides with distinct cellidentification sequences, a cell identification sequence associated withtwo or more cell identification oligonucleotides can be identified insequencing data and removed from subsequent processing. Such highernumber of cells can be loaded into microwells relative to the number ofmicrowells in a microwell cartridge or array.

Disclosed herein includes methods for sample identification. In someembodiments, the method comprises: contacting a first plurality of cellsand a second plurality of cells with two sample indexing compositionsrespectively, wherein each of the first plurality of cells and each ofthe second plurality of cells comprise one or more cellular components,wherein each of the two sample indexing compositions comprises acellular component binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular components, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of the two sample indexing compositions comprise differentsequences; barcoding the sample indexing oligonucleotides using aplurality of barcodes to create a plurality of barcoded sample indexingoligonucleotides, wherein each of the plurality of barcodes comprises acell label sequence, a barcode sequence (e.g., a molecular labelsequence), and a target-binding region, wherein the barcode sequences ofat least two barcodes of the plurality of barcodes comprise differentsequences, and wherein at least two barcodes of the plurality ofbarcodes comprise an identical cell label sequence; obtaining sequencingdata of the plurality of barcoded sample indexing oligonucleotides; andidentifying one or more cell label sequences that is each associatedwith two or more sample indexing sequences in the sequencing dataobtained; and removing the sequencing data associated with the one ormore cell label sequences that is each associated with two or moresample indexing sequences from the sequencing data obtained and/orexcluding the sequencing data associated with the one or more cell labelsequences that is each associated with two or more sample indexingsequences from subsequent analysis (e.g., single cell mRNA profiling, orwhole transcriptome analysis). In some embodiments, the sample indexingoligonucleotide comprises a barcode sequence (e.g., a molecular labelsequence), a binding site for a universal primer, or a combinationthereof.

For example, the method can be used to load 50000 or more cells(compared to 10000-20000 cells) using sample indexing. Sample indexingcan use oligonucleotide-conjugated cellular component binding reagents(e.g., antibodies) or cellular component binding reagents against acellular component (e.g., a universal protein marker) to label cellsfrom different samples with a unique sample index. When two or morecells from different samples, two or more cells from differentpopulations of cells of a sample, or two or more cells of a sample, arecaptured in the same microwell or droplet, the combined “cell” (orcontents of the two or more cells) can be associated with sampleindexing oligonucleotides with different sample indexing sequences (orcell identification oligonucleotides with different cell identificationsequences). The number of different populations of cells can bedifferent in different implementations. In some embodiments, the numberof different populations can be, or be about, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range betweenany two of these values. In some embodiments, the number of differentpopulations can be at least, or be at most, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, or 100. The number, or the averagenumber, of cells in each population can be different in differentimplementations. In some embodiments, the number, or the average number,of cells in each population can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a rangebetween any two of these values. In some embodiments, the number, or theaverage number, of cells in each population can be at least, or be atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or100. When the number, or the average number, of cells in each populationis sufficiently small (e.g., equal to, or fewer than, 50, 25, 10, 5, 4,3, 2, or 1 cells per population), the sample indexing composition forcell overloading and multiplet identification can be referred to as cellidentification compositions.

Cells of a sample can be divided into multiple populations by aliquotingthe cells of the sample into the multiple populations. A “cell”associated with more than one sample indexing sequence in the sequencingdata can be identified as a “multiplet” based on two or more sampleindexing sequences associated with one cell label sequence (e.g., a celllabel sequence of a barcode, such as a stochastic barcode) in thesequencing data. The sequencing data of a combined “cell” is alsoreferred to herein as a multiplet. A multiplet can be a doublet, atriplet, a quartet, a quintet, a sextet, a septet, an octet, a nonet, orany combination thereof. A multiplet can be any n-plet. In someembodiments, n is, or is about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or a range between any two of these values.In some embodiments, n is at least, or is at most, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

When determining expression profiles of single cells, two cells may beidentified as one cell and the expression profiles of the two cells maybe identified as the expression profile for one cell (referred to as adoublet expression profile). For example, when determining expressionprofiles of two cells using barcoding (e.g., stochastic barcoding), themRNA molecules of the two cells may be associated with barcodes havingthe same cell label. As another example, two cells may be associatedwith one particle (e.g., a bead). The particle can include barcodes withthe same cell label. After lysing the cells, the mRNA molecules in thetwo cells can be associated with the barcodes of the particle, thus thesame cell label. Doublet expression profiles can skew the interpretationof the expression profiles.

A doublet can refer to a combined “cell” associated with two sampleindexing oligonucleotides with different sample indexing sequences. Adoublet can also refer to a combined “cell” associated with sampleindexing oligonucleotides with two sample indexing sequences. A doubletcan occur when two cells associated with two sample indexingoligonucleotides of different sequences (or two or more cells associatedwith sample indexing oligonucleotides with two different sample indexingsequences) are captured in the same microwell or droplet, the combined“cell” can be associated with two sample indexing oligonucleotides withdifferent sample indexing sequences. A triplet can refer to a combined“cell” associated with three sample indexing oligonucleotides all withdifferent sample indexing sequences, or a combined “cell” associatedwith sample indexing oligonucleotides with three different sampleindexing sequences. A quartet can refer to a combined “cell” associatedwith four sample indexing oligonucleotides all with different sampleindexing sequences, or a combined “cell” associated with sample indexingoligonucleotides with four different sample indexing sequences. Aquintet can refer to a combined “cell” associated with five sampleindexing oligonucleotides all with different sample indexing sequences,or a combined “cell” associated with sample indexing oligonucleotideswith five different sample indexing sequences. A sextet can refer to acombined “cell” associated with six sample indexing oligonucleotides allwith different sample indexing sequences, or a combined “cell”associated with sample indexing oligonucleotides with six differentsample indexing sequences. A septet can refer to a combined “cell”associated with seven sample indexing oligonucleotides all withdifferent sample indexing sequences, or a combined “cell” associatedwith sample indexing oligonucleotides with seven different sampleindexing sequences. A octet can refer to a combined “cell” associatedwith eight sample indexing oligonucleotides all with different sampleindexing sequences, or a combined “cell” associated with sample indexingoligonucleotides with eight different sample indexing sequences. A nonetcan refer to a combined “cell” associated with nine sample indexingoligonucleotides all with different sample indexing sequences, or acombined “cell” associated with sample indexing oligonucleotides withnine different sample indexing sequences. A multiplet can occur when twoor more cells associated with two or more sample indexingoligonucleotides of different sequences (or two or more cells associatedwith sample indexing oligonucleotides with two or more different sampleindexing sequences) are captured in the same microwell or droplet, thecombined “cell” can be associated with sample indexing oligonucleotideswith two or more different sample indexing sequences.

As another example, the method can be used for multiplet identification,whether in the context of sample overloading or in the context ofloading cells onto microwells of a microwell array or generatingdroplets containing cells. When two or more cells are loaded into onemicrowell, the resulting data from the combined “cell” (or contents ofthe two or more cells) is a multiplet with aberrant gene expressionprofile. By using sample indexing, one can recognize some of thesemultiplets by looking for cell labels that are each associated with orassigned to two or more sample indexing oligonucleotides with differentsample indexing sequences (or sample indexing oligonucleotides with twoor more sample indexing sequences). With sample indexing sequence, themethods disclosed herein can be used for multiplet identification(whether in the context of sample overloading or not, or in the contextof loading cells onto microwells of a microwell array or generatingdroplets containing cells). In some embodiments, the method comprises:contacting a first plurality of cells and a second plurality of cellswith two sample indexing compositions respectively, wherein each of thefirst plurality of cells and each of the second plurality of cellscomprise one or more cellular components, wherein each of the two sampleindexing compositions comprises a cellular component binding reagentassociated with a sample indexing oligonucleotide, wherein the cellularcomponent binding reagent is capable of specifically binding to at leastone of the one or more cellular components, wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of the two sample indexing compositions comprisedifferent sequences; barcoding the sample indexing oligonucleotidesusing a plurality of barcodes to create a plurality of barcoded sampleindexing oligonucleotides, wherein each of the plurality of barcodescomprises a cell label sequence, a barcode sequence (e.g., a molecularlabel sequence), and a target-binding region, wherein barcode sequencesof at least two barcodes of the plurality of barcodes comprise differentsequences, and wherein at least two barcodes of the plurality ofbarcodes comprise an identical cell label sequence; obtaining sequencingdata of the plurality of barcoded sample indexing oligonucleotides; andidentifying one or more multiplet cell label sequences that is eachassociated with two or more sample indexing sequences in the sequencingdata obtained.

The number of cells that can be loaded onto microwells of a microwellcartridge or into droplets generated using a microfluidics device can belimited by the multiplet rate. Loading more cells can result in moremultiplets, which can be hard to identify and create noise in the singlecell data. With sample indexing, the method can be used to moreaccurately label or identify multiplets and remove the multiplets fromthe sequencing data or subsequent analysis. Being able to identifymultiplets with higher confidence can increase user tolerance for themultiplet rate and load more cells onto each microwell cartridge orgenerating droplets with at least one cell each.

In some embodiments, contacting the first plurality of cells and thesecond plurality of cells with the two sample indexing compositionsrespectively comprises: contacting the first plurality of cells with afirst sample indexing compositions of the two sample indexingcompositions; and contacting the first plurality of cells with a secondsample indexing compositions of the two sample indexing compositions.The number of pluralities of cells and the number of pluralities ofsample indexing compositions can be different in differentimplementations. In some embodiments, the number of pluralities of cellsand/or sample indexing compositions can be, or be about, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 10000, 100000, 1000000, or a number or a rangebetween any two of these values. In some embodiments, the number ofpluralities of cells and/or sample indexing compositions can be atleast, or be at most, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000,100000, or 1000000. The number of cells can be different in differentimplementations. In some embodiments, the number, or the average number,of cells can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,10000, 100000, 1000000, or a number or a range between any two of thesevalues. In some embodiments, the number, or the average number, or cellscan be at least, or be at most, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,10000, 100000, or 1000000.

In some embodiments, the method comprises: removing unbound sampleindexing compositions of the two sample indexing compositions. Removingthe unbound sample indexing compositions can comprise washing cells ofthe first plurality of cells and the second plurality of cells with awashing buffer. Removing the unbound sample indexing compositions cancomprise selecting cells bound to at least one cellular componentbinding reagent of the two sample indexing compositions using flowcytometry. In some embodiments, the method comprises: lysing the one ormore cells from each of the plurality of samples.

In some embodiments, the sample indexing oligonucleotide is configuredto be detachable or non-detachable from the cellular component bindingreagent. The method can comprise detaching the sample indexingoligonucleotide from the cellular component binding reagent. Detachingthe sample indexing oligonucleotide can comprise detaching the sampleindexing oligonucleotide from the cellular component binding reagent byUV photocleaving, chemical treatment (e.g., using reducing reagent, suchas dithiothreitol), heating, enzyme treatment, or any combinationthereof.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises: amplifying the plurality ofbarcoded sample indexing oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded sample indexingoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of barcode sequence (e.g., themolecular label sequence) and at least a portion of the sample indexingoligonucleotide. In some embodiments, obtaining the sequencing data ofthe plurality of barcoded sample indexing oligonucleotides can compriseobtaining sequencing data of the plurality of amplicons. Obtaining thesequencing data comprises sequencing at least a portion of the barcodesequence and at least a portion of the sample indexing oligonucleotide.In some embodiments, identifying the sample origin of the at least onecell comprises identifying sample origin of the plurality of barcodedtargets based on the sample indexing sequence of the at least onebarcoded sample indexing oligonucleotide.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes to create the plurality of barcodedsample indexing oligonucleotides comprises stochastically barcoding thesample indexing oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded sampleindexing oligonucleotides.

In some embodiments, the method includes: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label sequence, and wherein at least two barcodes ofthe plurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can include: contacting copies of thetargets with target-binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets.

In some embodiments, the method comprises: prior to obtaining thesequencing data of the plurality of barcoded targets, amplifying thebarcoded targets to create a plurality of amplified barcoded targets.Amplifying the barcoded targets to generate the plurality of amplifiedbarcoded targets can comprise: amplifying the barcoded targets bypolymerase chain reaction (PCR). Barcoding the plurality of targets ofthe cell using the plurality of barcodes to create the plurality ofbarcoded targets can comprise stochastically barcoding the plurality oftargets of the cell using a plurality of stochastic barcodes to create aplurality of stochastically barcoded targets.

In some embodiments, the method for cell identification comprise:contacting a first plurality of one or more cells and a second pluralityof one or more cells with two cell identification compositionsrespectively, wherein each of the first plurality of one or more cellsand each of the second plurality of one or more cells comprise one ormore cellular components, wherein each of the two cell identificationcompositions comprises a cellular component binding reagent associatedwith a cell identification oligonucleotide, wherein the cellularcomponent binding reagent is capable of specifically binding to at leastone of the one or more cellular components, wherein the cellidentification oligonucleotide comprises a cell identification sequence,and wherein cell identification sequences of the two cell identificationcompositions comprise different sequences; barcoding the cellidentification oligonucleotides using a plurality of barcodes to createa plurality of barcoded cell identification oligonucleotides, whereineach of the plurality of barcodes comprises a cell label sequence, abarcode sequence (e.g., a molecular label sequence), and atarget-binding region, wherein the barcode sequences of at least twobarcodes of the plurality of barcodes comprise different sequences, andwherein at least two barcodes of the plurality of barcodes comprise anidentical cell label sequence; obtaining sequencing data of theplurality of barcoded cell identification oligonucleotides; andidentifying one or more cell label sequences that is each associatedwith two or more cell identification sequences in the sequencing dataobtained; and removing the sequencing data associated with the one ormore cell label sequences that is each associated with two or more cellidentification sequences from the sequencing data obtained and/orexcluding the sequencing data associated with the one or more cell labelsequences that is each associated with two or more cell identificationsequences from subsequent analysis (e.g., single cell mRNA profiling, orwhole transcriptome analysis). In some embodiments, the cellidentification oligonucleotide comprises a barcode sequence (e.g., amolecular label sequence), a binding site for a universal primer, or acombination thereof.

A multiplet (e.g., a doublet, triplet, etc) can occur when two or morecells associated with two or more cell identification oligonucleotidesof different sequences (or two or more cells associated with cellidentification oligonucleotides with two or more different cellidentification sequences) are captured in the same microwell or droplet,the combined “cell” can be associated with cell identificationoligonucleotides with two or more different cell identificationsequences.

Cell identification compositions can be used for multipletidentification, whether in the context of cell overloading or in thecontext of loading cells onto microwells of a microwell array orgenerating droplets containing cells. When two or more cells are loadedinto one microwell, the resulting data from the combined “cell” (orcontents of the two or more cells) is a multiplet with aberrant geneexpression profile. By using cell identification, one can recognize someof these multiplets by looking for cell labels (e.g., cell labels ofbarcodes, such as stochastic barcodes) that are each associated with orassigned to two or more cell identification oligonucleotides withdifferent cell identification sequences (or cell identificationoligonucleotides with two or more cell identification sequences). Withcell identification sequence, the methods disclosed herein can be usedfor multiplet identification (whether in the context of sampleoverloading or not, or in the context of loading cells onto microwellsof a microwell array or generating droplets containing cells). In someembodiments, the method comprises: contacting a first plurality of oneor more cells and a second plurality of one or more cells with two cellidentification compositions respectively, wherein each of the firstplurality of one or more cells and each of the second plurality of oneor more cells comprise one or more cellular components, wherein each ofthe two cell identification compositions comprises a cellular componentbinding reagent associated with a cell identification oligonucleotide,wherein the cellular component binding reagent is capable ofspecifically binding to at least one of the one or more cellularcomponents, wherein the cell identification oligonucleotide comprises acell identification sequence, and wherein cell identification sequencesof the two cell identification compositions comprise differentsequences; barcoding the cell identification oligonucleotides using aplurality of barcodes to create a plurality of barcoded cellidentification oligonucleotides, wherein each of the plurality ofbarcodes comprises a cell label sequence, a barcode sequence (e.g., amolecular label sequence), and a target-binding region, wherein barcodesequences of at least two barcodes of the plurality of barcodes comprisedifferent sequences, and wherein at least two barcodes of the pluralityof barcodes comprise an identical cell label sequence; obtainingsequencing data of the plurality of barcoded cell identificationoligonucleotides; and identifying one or more multiplet cell labelsequences that is each associated with two or more cell identificationsequences in the sequencing data obtained.

The number of cells that can be loaded onto microwells of a microwellcartridge or into droplets generated using a microfluidics device can belimited by the multiplet rate. Loading more cells can result in moremultiplets, which can be hard to identify and create noise in the singlecell data. With cell identification, the method can be used to moreaccurately label or identify multiplets and remove the multiplets fromthe sequencing data or subsequent analysis. Being able to identifymultiplets with higher confidence can increase user tolerance for themultiplet rate and load more cells onto each microwell cartridge orgenerating droplets with at least one cell each.

In some embodiments, contacting the first plurality of one or more cellsand the second plurality of one or more cells with the two cellidentification compositions respectively comprises: contacting the firstplurality of one or more cells with a first cell identificationcompositions of the two cell identification compositions; and contactingthe first plurality of one or more cells with a second cellidentification compositions of the two cell identification compositions.The number of pluralities of cell identification compositions can bedifferent in different implementations. In some embodiments, the numberof cell identification compositions can be, or be about, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 10000, 100000, 1000000, or a number or a rangebetween any two of these values. In some embodiments, the number of cellidentification compositions can be at least, or be at most, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 10000, 100000, or 1000000. The number, oraverage number, of cells in each plurality of one or more cells can bedifferent in different implementations. In some embodiments, the number,or average number, of cells in each plurality of one or more cells canbe, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000,100000, 1000000, or a number or a range between any two of these values.In some embodiments, the number, or average number, of cells in eachplurality of one or more cells can be at least, or be at most, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 10000, 100000, or 1000000.

In some embodiments, the method comprises: removing unbound cellidentification compositions of the two cell identification compositions.Removing the unbound cell identification compositions can comprisewashing cells of the first plurality of one or more cells and the secondplurality of one or more cells with a washing buffer. Removing theunbound cell identification compositions can comprise selecting cellsbound to at least one cellular component binding reagent of the two cellidentification compositions using flow cytometry. In some embodiments,the method comprises: lysing the one or more cells from each of theplurality of samples.

In some embodiments, the cell identification oligonucleotide isconfigured to be detachable or non-detachable from the cellularcomponent binding reagent. The method can comprise detaching the cellidentification oligonucleotide from the cellular component bindingreagent. Detaching the cell identification oligonucleotide can comprisedetaching the cell identification oligonucleotide from the cellularcomponent binding reagent by UV photocleaving, chemical treatment (e.g.,using reducing reagent, such as dithiothreitol), heating, enzymetreatment, or any combination thereof.

In some embodiments, barcoding the cell identification oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the cell identification oligonucleotides to generatebarcodes hybridized to the cell identification oligonucleotides; andextending the barcodes hybridized to the cell identificationoligonucleotides to generate the plurality of barcoded cellidentification oligonucleotides. Extending the barcodes can compriseextending the barcodes using a DNA polymerase to generate the pluralityof barcoded cell identification oligonucleotides. Extending the barcodescan comprise extending the barcodes using a reverse transcriptase togenerate the plurality of barcoded cell identification oligonucleotides.

In some embodiments, the method comprises: amplifying the plurality ofbarcoded cell identification oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded cell identificationoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of barcode sequence (e.g., themolecular label sequence) and at least a portion of the cellidentification oligonucleotide. In some embodiments, obtaining thesequencing data of the plurality of barcoded cell identificationoligonucleotides can comprise obtaining sequencing data of the pluralityof amplicons. Obtaining the sequencing data comprises sequencing atleast a portion of the barcode sequence and at least a portion of thecell identification oligonucleotide. In some embodiments, identifyingthe sample origin of the at least one cell comprises identifying sampleorigin of the plurality of barcoded targets based on the cellidentification sequence of the at least one barcoded cell identificationoligonucleotide.

In some embodiments, barcoding the cell identification oligonucleotidesusing the plurality of barcodes to create the plurality of barcoded cellidentification oligonucleotides comprises stochastically barcoding thecell identification oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded cellidentification oligonucleotides.

Determining Cellular Component-Cellular Component Interactions

Disclosed herein include methods, for example multiplexed proximityligation methods, for determining interactions between cellularcomponents. Such methods, kits and systems can be used in combinationwith any suitable methods, kits and systems disclosed herein, forexample the methods, kits and systems for measuring cellular componentexpression level (such as protein expression level) using cellularcomponent binding reagents associated with oligonucleotides.

The methods disclosed herein can be used to detect, identify, determine,and/or measure interactions between one or multiple cellular components(for example, proteins). For example, the methods can be used to detect,identify, determine, and/or measure interactions between two, three,four, five, six, seven, eight, nine, ten, twenty, thirty, forty, fifty,one hundred, two hundred, three hundred, or more pairs of cellularcomponents. The cellular component targets in one pair of cellularcomponents targets can overlap with the cellular component targets inanother pair of cellular component targets. For example, the first pairof cellular component targets can include protein A and protein B, andthe second pair of cellular component targets can include protein A andprotein C.

In some embodiments, the method uses cellular component binding reagentsassociated with interaction determination oligonucleotides to determineinteractions between targets of the cellular component binding reagents(referred to herein as cellular component targets). For example, themethod can use antibody-oligonucleotide conjugates to detectprotein-protein interactions in single cells. The antibodies can behomodimers or heterodimers. Each cellular component binding reagent canbe associated with (e.g., attached to or bind to) an interactiondetermination oligonucleotide containing a unique interactiondetermination sequence. For example, each antibody can be conjugated toan oligonucleotide containing a unique barcode sequence. Determining thepairs of interaction determination oligonucleotides that are ligatedtogether can reveal the cellular component targets that interact witheach other. For example, determining the barcode pairs that are ligatedtogether can reveal the proteins interact with each other. This methodcan also be used in conjunction with single cell mRNA sequencingmethods, such as well-based and droplet-based single cell mRNAsequencing methods.

In some embodiments, the method can utilize linear amplification togenerate amplicons for sequencing. The method may not generate acircular template and use rolling circle amplification to generatesufficient template for downstream detection by fluorescent probes, orother methods. The method can be used to determine interactions betweencellular component targets in single cells. For example, the method canbe used to detect hundreds of cellular component-cellular componentinteractions (e.g., protein-protein interactions) across thousands totens of thousands of single cells and uses sequencing as readouts.Interactions between the same proteins or cellular component targets canalso be determined. The method does not rely on fluorescence andquantitative PCR (qPCR) as the readouts, which require bulk RNA asinput, can only process few cells (e.g., less than 100 cells) at a time,and can detect a limited amount of cellular component-cellular componentinteractions at a time. The multiplexed method can utilize barcodedoligonucleotides associated with or conjugated to antibodies or cellularcomponent binding reagents can be used to determine cellularcomponent-cellular component interactions in a large number of singlecells in a high throughput manner.

Furthermore, the method can also be used with other single cell RNAsequencing methods. For example, one single workflow can be used todetermine mRNA expression levels, protein expression levels (or levelsof cellular component targets), and/or protein-protein interactions (orinteractions between cellular components). As a result, in a singleexperiment, data about mRNA expression, protein expression, andprotein-protein interactions from in a single cell (or multiple singlecells) can be obtained.

In some embodiments, the method can be used to detect cellularcomponent-cellular component interactions between two cells andintracellular cellular component-cellular component interactions. Forexample, the method can be used to detect protein-protein interactionsbetween two cells, as well as intracellular protein-proteininteractions. The method may require additional cell preparation beforeantibody staining or incubation (e.g., cell fixation to allow binding ofantibodies to intracellular proteins or targets).

FIGS. 14A-14F show a schematic illustration of an exemplary workflow ofdetermining interactions between cellular components (e.g., proteins)using a pair of interaction determination compositions. The method canutilize two types of antibody-oligonucleotide compositions 1404 a, 1404b (e.g., antibody-oligonucleotide conjugates) illustrated in FIG. 14A.The two types of antibody-oligonucleotide compositions 1404 a, 1404 bcan include two identical or different antibodies 1408 a, 1408 bassociated with two types of oligonucleotides 1412 a, 1412 b. Theassociation (e.g., conjugation) can be reversible or non-reversible(e.g., cleavable or non-cleavable). One type of oligonucleotides 1412 a(referred to herein as the type 1 oligonucleotide 1412 a) can have auniversal sequencing region 1416. In some embodiments, the universalsequencing region 1416 can be common to all antibody-oligonucleotidecompositions for amplification with a universal primer. The other typeof oligonucleotides 1412 b (referred to herein as the type 2oligonucleotide 1412 b) can have a poly(dA) sequence 1420 forhybridization to barcodes, such as stochastic barcodes. For example, thepoly(dA) sequence 1420 of the type two oligonucleotide 1412 b canhybridize to stochastic barcodes associated with a bead (e.g.,immobilized on the bead, partially immobilized on the bead, releasablyenclosed in the bead, partially enclosed in the bead, or any combinationthereof). Both oligonucleotide types 1412 a, 1412 b also include abarcode sequence 1424 a, 1424 b unique to each antibody 1404 a, 1404 b.The two oligonucleotide types 1412 a, 1412 b can include differentsequences 1428 a, 1428 b for hybridization to a bridge oligo 1432illustrated in FIG. 14B. Although the type one oligonucleotide 1412 aand the type two oligonucleotide 1412 b are shown to be associated withthe antibodies 1408 a, 1408 b in the 5′ to 3′ direction and the 3′ to 5′direction, they are illustrative only and are not intended to belimiting. In some embodiments, the type one oligonucleotide 1412 a andthe type two oligonucleotide 1412 b can be associated with theantibodies 1408 a, 1408 b in the 3′ to 5′ direction and the 5′ to 3′direction.

Referring to FIG. 14B, the bridge oligonucleotide 1432 can bind to thebridge oligonucleotide hybridization regions 1428 a, 1428 b to bring theantibody-oligonucleotide compositions 1404 a, 1404 b together forligation. The sequences of the bridge oligonucleotide hybridizationregions 1428 a, 1428 b can be different for each composition 1404 a,1404 b. In some embodiments, a different bridge oligonucleotide 1432 canbe used for each antibody pair.

The methods can be based on bringing together pairs ofantibody-oligonucleotide compositions 1404 a, 1404 b so that theassociated oligonucleotides 1412 a, 1412 b can be joined together orligated to each other, and later captured by barcodes (e.g., stochasticbarcodes) associated with beads for sequencing. The oligonucleotides1412 a, 1412 b can be ligated to each other if the targets 1436 a, 1436b of the antibodies 1408 a, 1408 b are within a certain thresholddistance or range of each other, such as 30-40 nm.

The multiplexed method can utilize barcoded oligonucleotides associatedwith or conjugated to antibodies or cellular component binding reagentsto determine protein-protein interactions, or interactions betweencellular components, in a large number of single cells in a highthroughput manner. In some embodiments, the interactions betweencellular components in, or in about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 10⁵, 10⁶, 10⁷, 10⁸,10⁹, or a number or a range between any two of these values, cells canbe determined in a cell workflow. In some embodiments, the interactionsbetween cellular components in at least, or in at most, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or a number or a range between any two of thesevalues, cells can be determined in a cell workflow.

Staining

To perform the assay, cells can be first stained withantibody-oligonucleotide compositions 1404 a, 1404 b or pairs ofantibody-oligonucleotide compositions 1404 a, 1404 b (or cellularcomponent binding reagent-oligonucleotide compositions). The number ofantibody-oligonucleotide compositions 1404 a, 1404 b (or cellularcomponent binding reagents) can be different in differentimplementations. In some embodiments, the number ofantibody-oligonucleotide compositions 1404 a, 1404 b can be, or beabout, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or a rangebetween any two of these values. In some embodiments, the number ofantibody-oligonucleotide compositions 1404 a, 1404 b can be at least, orbe at most, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.

The antibodies 1408 a, 1408 b (or cellular component binding reagents)of different compositions 1404 a, 1404 b can be associated with or bindto interaction determination oligonucleotides 1412 a, 1412 b withdifferent or identical barcode sequences 1424 a, 1424 b. In someembodiments, the number of antibodies 1408 a, 1408 b of differentcompositions 1404 a, 1404 b associated with or bind to interactiondetermination oligonucleotides 1412 a, 1412 b with different barcodesequences or identical barcode sequences 1424 a, 1424 b can be, or beabout, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or a rangebetween any two of these values. In some embodiments, the number ofantibodies 1408 a, 1408 b of different compositions 1404 a, 1404 bassociated with or bind to interaction determination oligonucleotides1412 a, 1412 b with different barcode sequences or identical barcodesequences 1424 a, 1424 b can be at least, or be at most, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000. In some embodiments, the percentage ofantibodies 1408 a, 1408 b of different compositions 1404 a, 1404 bassociated with or bind to interaction determination oligonucleotides1412 a, 1412 b with different barcode sequences or identical barcodesequences 1424 a, 1424 b can be, or be about, 0.1%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 20%, 33%, 40%, 50%, 60%, 70%, 80%, 90%, 99.9%,99.99%, 100%, or a number or a range between any two of these values. Insome embodiments, the percentage of antibodies 1408 a, 1408 b ofdifferent compositions 1404 a, 1404 b associated with or bind tointeraction determination oligonucleotides 1412 a, 1412 b with differentbarcode sequences or identical barcode sequences 1424 a, 1424 b can beat least, or be at most, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 33%, 40%, 50%, 60%, 70%, 80%, 90%, 99.9%, 99.99%, or 100%.

Some, all, or none of the antibodies 1408 a, 1408 b (or cellularcomponent binding reagents) can be associated with or bind tointeraction determination oligonucleotides 1412 a, 1412 b having bridgeoligonucleotide hybridization regions 1428 a, 1428 b with identicalsequence. In some embodiments, the number of antibodies 1408 a, 1408 bassociated with or bind to interaction determination oligonucleotides1412 a, 1412 b having bridge oligonucleotide hybridization regions 1428a, 1428 b with identical sequence or different sequences can be, or beabout, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or arange between any two of these values. In some embodiments, the numberof antibodies 1408 a, 1408 b associated with or bind to interactiondetermination oligonucleotides 1412 a, 1412 b having bridgeoligonucleotide hybridization regions 1428 a, 1428 b with identicalsequence or different sequences can be at least, or be at most, 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, or 1000. In some embodiments, thepercentage of antibodies 1408 a, 1408 b associated with or bind tointeraction determination oligonucleotides 1412 a, 1412 b having bridgeoligonucleotide hybridization regions 1428 a, 1428 b with identicalsequence or different sequences can be, or be about, 0%, 0.1%, 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 33%, 40%, 50%, 60%, 70%, 80%, 90%,99.9%, 99.99%, 100%, or a number or a range between any two of thesevalues. In some embodiments, the percentage of antibodies 1408 a, 1408 bassociated with or bind to interaction determination oligonucleotides1412 a, 1412 b having bridge oligonucleotide hybridization regions 1428a, 1428 b with identical sequence or different sequences can be atleast, or be at most, 0%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 33%, 40%, 50%, 60%, 70%, 80%, 90%, 99.9%, 99.99%, or 100%.

Some, all, or none of the antibodies 1408 a (or cellular componentbinding reagents) can be associated with or bind to interactiondetermination oligonucleotides 1412 a having universal sequencingregions with identical sequence or different sequences. In someembodiments, the number of antibodies 1408 a associated with or bind tointeraction determination oligonucleotides 1412 a having universalsequencing regions 1416 a with identical sequence or different sequencescan be, or be about, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or anumber or a range between any two of these values. In some embodiments,the number of antibodies 1408 a associated with or bind to interactiondetermination oligonucleotides 1412 a having universal sequencingregions 1416 a with identical sequence or different sequences can be atleast, or be at most, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000. Insome embodiments, the percentage of antibodies 1408 a associated with orbind to interaction determination oligonucleotides 1412 a havinguniversal sequencing regions 1416 a with identical sequence or differentsequences can be, or be about, 0%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 20%, 33%, 40%, 50%, 60%, 70%, 80%, 90%, 99.9%, 99.99%, 100%, ora number or a range between any two of these values. In someembodiments, the number of antibodies 1408 a associated with or bind tointeraction determination oligonucleotides 1412 a having universalsequencing regions 1416 a with identical sequence or different sequencescan be at least, or be at most, 0%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 33%, 40%, 50%, 60%, 70%, 80%, 90%, 99.9%, 99.99%, or100%.

The number of pairs of antibody-oligonucleotide compositions 1404 a,1404 b (cellular component binding reagent-oligonucleotide compositions)can be different in different implementations. In some embodiments, thenumber of pairs of antibody-oligonucleotide compositions 1404 a, 1404 bcan be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or anumber or a range between any two of these values. In some embodiments,the number of pairs of antibody-oligonucleotide compositions 1404 a,1404 b can be at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, or 1000.

The antibodies 1408 a, 1408 b (or cellular component binding reagents)of different pairs of compositions 1404 a, 1404 b can be associated withor bind to interaction determination oligonucleotides 1412 a, 1412 bwith different or the identical barcode sequences 1424 a, 1424 b. Insome embodiments, the number of antibodies 1408 a, 1408 b of differentpairs of compositions 1404 a, 1404 b associated with or bind tointeraction determination oligonucleotides 1412 a, 1412 b with differentbarcode sequences or identical barcode sequences 1424 a, 1424 b can be,or be about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or arange between any two of these values. In some embodiments, the numberof antibodies 1408 a, 1408 b of different pairs of compositions 1404 a,1404 b associated with or bind to interaction determinationoligonucleotides 1412 a, 1412 b with different barcode sequences oridentical barcode sequences 1424 a, 1424 b can be at least, or be atmost, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000. In some embodiments,the percentage of antibodies 1408 a, 1408 b of different pairs ofcompositions 1404 a, 1404 b associated with or bind to interactiondetermination oligonucleotides 1412 a, 1412 b with different barcodesequences or identical barcode sequences 1424 a, 1424 b can be, or beabout, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 33%, 40%,50%, 60%, 70%, 80%, 90%, 99.9%, 99.99%, 100%, or a number or a rangebetween any two of these values. In some embodiments, the percentage ofantibodies 1408 a, 1408 b of different pairs of compositions 1404 a,1404 b associated with or bind to interaction determinationoligonucleotides 1412 a, 1412 b with different barcode sequences oridentical barcode sequences 1424 a, 1424 b can be at least, or be atmost, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 33%, 40%, 50%,60%, 70%, 80%, 90%, 99.9%, 99.99%, or 100%.

Some, all, or none of the antibodies 1408 a, 1408 b (or cellularcomponent binding reagents) of different pairs of compositions 1404 a,1404 b can be associated with or bind to interaction determinationoligonucleotides 1412 a, 1412 b with identical bridge oligonucleotidehybridization regions. In some embodiments, the number of antibodies1408 a, 1408 b of different pairs of compositions 1404 a, 1404 bassociated with or bind to interaction determination oligonucleotides1412 a, 1412 b having bridge oligonucleotide hybridization regions 1428a, 1428 b with identical sequence or different sequences can be, or beabout, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or arange between any two of these values. In some embodiments, the numberof antibodies 1408 a, 1408 b of different pairs of compositions 1404 a,1404 b associated with or bind to interaction determinationoligonucleotides 1412 a, 1412 b having bridge oligonucleotidehybridization regions 1428 a, 1428 b with identical sequence ordifferent sequences can be at least, or be at most, 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000. In some embodiments, the % of antibodies1408 a, 1408 b of different pairs of compositions 1404 a, 1404 bassociated with or bind to interaction determination oligonucleotides1412 a, 1412 b having bridge oligonucleotide hybridization regions 1428a, 1428 b with identical sequence or different sequences can be, or beabout, 0%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 33%, 40%,50%, 60%, 70%, 80%, 90%, 99.9%, 99.99%, 100%, or a number or a rangebetween any two of these values. In some embodiments, the percentage ofantibodies 1408 a, 1408 b of different pairs of compositions 1404 a,1404 b associated with or bind to interaction determinationoligonucleotides 1412 a, 1412 b having bridge oligonucleotidehybridization regions 1428 a, 1428 b with identical sequence ordifferent sequences can be at least, or be at most, 0%, 0.1%, 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 33%, 40%, 50%, 60%, 70%, 80%, 90%,99.9%, 99.99%, or 100%.

Some, all, or none of the antibodies 1408 a (or cellular componentbinding reagents) of different compositions 1404 a can be associatedwith or bind to interaction determination oligonucleotides 1412 a havinguniversal sequencing regions with identical sequence or differentsequences. In some embodiments, the number of antibodies 1408 a ofdifferent compositions 1404 a associated with or bind to interactiondetermination oligonucleotides 1412 a having universal sequencingregions 1416 a with identical sequence or different sequences can be, orbe about, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or arange between any two of these values. In some embodiments, the numberof antibodies 1408 a of different compositions 1404 a associated with orbind to interaction determination oligonucleotides 1412 a havinguniversal sequencing regions 1416 a with identical sequence or differentsequences can be at least, or be at most, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, or 1000. In some embodiments, the percentage of antibodies1408 a of different compositions 1404 a associated with or bind tointeraction determination oligonucleotides 1412 a having universalsequencing regions 1416 a with identical sequence or different sequencescan be, or be about, 0%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 33%, 40%, 50%, 60%, 70%, 80%, 90%, 99.9%, 99.99%, 100%, or a numberor a range between any two of these values. In some embodiments, thepercentage of antibodies 1408 a of different compositions 1404 aassociated with or bind to interaction determination oligonucleotides1412 a having universal sequencing regions 1416 a with identicalsequence or different sequences can be at least, or be at most, 0%,0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 33%, 40%, 50%, 60%,70%, 80%, 90%, 99.9%, 99.99%, or 100%.

The number of targets 1436 a, 1436 b can be different in differentimplementations, such as 2 to at least 50-100 protein targets. In someembodiments, the number of targets 1436 a, 1436 b can be, or be about,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, or a number or a range betweenany two of these values. In some embodiments, the number of targets 1436a, 1436 b can be at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, or 1000.

Ligation

After washing away unbound compositions 1404 a, 1404 b, a bridgeoligonucleotide 1432 can be added. Pairs made of type 1 and type 2compositions 1404 a, 1404 b can be linked together via the bridgeoligonucleotide 1432 which can hybridize to bridge oligonucleotidehybridization regions. For example, pairs made of type 1 and type 2compositions 1404 a, 1404 b can be linked together via the bridgeoligonucleotide 1432 which can hybridize to bridge oligonucleotidehybridization regions if the corresponding protein targets 1436 a, 1436b are within a threshold distance, such as 30-40 nm. DNA ligase can thenbe added to ligate the two oligonucleotides 1412 a, 1412 b to form aligated oligonucleotide 1412, resulting in the compositions 1436 a, 1436b being ligated together. For example, the bridge oligonucleotide 1432can make the ligation region double-stranded to facilitate ligation. Thebridge oligonucleotide 1432 can bind to the hybridization regions 1428a, 1428 b on each oligonucleotide 1412 a, 1412 b, which allows a DNAligase to ligate the two oligonucleotides 1412 a, 1412 b together.

The threshold distance can be different in different implementations. Insome embodiments, the threshold distance can be, or be about, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000 nm, or a number or a range betweenany two of these values. For example, the distance can be in the range30-40 nm. In some embodiments, the threshold distance can be at least,or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nm. Thethreshold distance can be affected by the lengths of theoligonucleotides 1412 a, 1412 b, locations of the associations orattachments of the oligonucleotides 1412 a, 1412 b to the antibodies1408 a, 1408 b, the size of the targets 1436 a, 1436 b, or anycombination thereof.

Cell Lysis and Ligated Oligonucleotide Hybridization to Barcode

Then, cells can be lysed to allow capture of the ligated oligonucleotide1412 as illustrated in FIG. 14C. For example, a bead 1438 with a barcode1440 (e.g., a stochastic barcode) having a poly(dT) region 1444 cancapture the ligated oligonucleotide 1412 via the poly(dA) region. Thebead 1438 can additional include labels, such as cell and/or molecularlabels 1448 and a universal sequence 1452 for subsequence amplification.

Reverse Transcription

Reverse transcription can then performed to generate the complementarysequence 1456 of the ligated oligonucleotide 1412 shown in FIG. 14D. Forexample, a Moloney Murine Leukemia Virus (MMLV)-based or Taq-basedreverse transcriptase can be used to generate the complementary sequenceof the ligated oligonucleotide 1412. The MMLV-based reversetranscriptase has a strand-displacing mechanism to displace the bridgeoligonucleotide 1432 during generation of the complementary strand. TheTaq-based reverse transcriptase has 5′ to 3′ exonuclease activity toremove the bridge oligonucleotide 1432.

Library Preparation

After reverse transcription, a sequencing library can be prepared, forexample, following standard protocols for single cell mRNA sequencing.Referring to FIG. 14E, the complementary sequence 1456 (or a reversecomplement thereof 1456 r) can be amplified via the universal sequence1452 (e.g., the universal P5 sequence or a partial universal P5sequence) on the barcode 1440 and the universal sequencing region 1416(e.g., the universal N1 sequence) of the type oneantibody-oligonucleotide composition 1404 a. The superscript r denotes areverse complement. The first round of PCR (referred to herein as PCR1)can use universal primers (e.g., primers with P5 and N1 sequences, orreverse complements thereof) to generate full length (or close to fulllength) amplicons that correspond to ligated oligonucleotide 1456 r withone or more labels and the universal sequence.

The same primers can be used for the second around of PCR amplificationreferred to herein as PCR2), or the N1 primer can be replaced by an N2primer that binds to both bridge oligonucleotide hybridization regions1428 a, 1428 b for further specificity as illustrated in FIG. 14F. Whenthis N2 primer is used, a majority of ligated oligonucleotide 1456 rwith both the universal sequencing region 1416 and the universalsequence 1452 can be amplified. In some embodiments, when this N2 primeris used for the second round of PCR, a majority of ligatedoligonucleotide 1456 r with both the universal sequencing region 1416and the universal sequence 1452 can be amplified.

Using Pairs of Interaction Determination Compositions to DetermineInteractions Between Cellular Components

Disclosed herein include systems, methods, and kits for determiningprotein-protein interactions. In some embodiments, the method comprises:contacting a cell with a first pair of interaction determinationcompositions 1404 a, 1404 b illustrated in FIG. 14A. The cell cancomprise a first protein target 1436 a and a second protein target 1436b. Each of the first pair of interaction determination compositions 1404a, 1404 b comprises a protein binding reagent 1408 a, 1408 b associatedwith an interaction determination oligonucleotide 1412 a, 1412 b. Theprotein binding reagent 1408 a, 1408 b of one of the first pair ofinteraction determination compositions is capable of specificallybinding to the first protein target 1436 a, and the protein bindingreagent 1408 b of the other of the first pair of interactiondetermination compositions is capable of specifically binding to thesecond protein target 1436 b. The interaction determinationoligonucleotide 1412 a, 1412 b comprises an interaction determinationsequence 1424 a, 1434 b and a bridge oligonucleotide hybridizationregion 1428 a, 1428 b. The interaction determination sequences 1424 a,1424 b of the first pair of interaction determination compositions 1404a, 1404 b can comprise different sequences.

Referring to FIG. 14B, the method can include ligating the interactiondetermination oligonucleotides 1412 a, 1412 b of the first pair ofinteraction determination compositions 1404 a, 1404 b using a bridgeoligonucleotide 1432 to generate a ligated interaction determinationoligonucleotide 1412. The bridge oligonucleotide 1432 can comprise twohybridization regions capable of specifically binding to the bridgeoligonucleotide hybridization regions 1428 a, 1428 b of the first pairof interaction determination compositions 1404 a, 1404 b

Referring to FIG. 14C, the method can include barcoding the ligatedinteraction determination oligonucleotide 1412 using a plurality ofbarcodes 1440 to create a plurality of barcoded interactiondetermination oligonucleotides 1456. Each of the plurality of barcodes1440 can comprise a barcode sequence 1448 and a capture sequence 1444.The method can include obtaining sequencing data of the plurality ofbarcoded interaction determination oligonucleotides 1456. The method caninclude determining an interaction between the first and second proteintargets 1436 a, 1436 b based on the association of the interactiondetermination sequences 1424 a, 1424 b of the first pair of interactiondetermination compositions 1404 a, 1404 b in the obtained sequencingdata.

Disclosed herein include systems, methods, and kits for determininginteractions between cellular component targets. In some embodiments,the method comprises: contacting a cell with a first pair of interactiondetermination compositions 1404 a, 1404 b illustrated in FIG. 14A. Thecell can comprise a first cellular component target and a secondcellular component target (e.g., a first protein target 1436 a and asecond protein target 1436 b). Each of the first pair of interactiondetermination compositions can comprise a cellular component bindingreagent (e.g., the antibody 1408 a, 1408 b) associated with aninteraction determination oligonucleotide 1412 a, 1412 b. The cellularcomponent binding reagent of one of the first pair of interactiondetermination compositions 1404 a, 1404 b is capable of specificallybinding to the first cellular component target (e.g., the protein target1436 a), and the cellular component binding reagent of the other 1404 bof the first pair of interaction determination compositions is capableof specifically binding to the second cellular component target (e.g.,the protein target 1436 b). The interaction determinationoligonucleotide 1412 a, 1412 b can comprise an interaction determinationsequence 1424 a, 1424 b and a bridge oligonucleotide hybridizationregion 1428 a, 1428 b. The interaction determination sequences 1424 a,1424 b of the first pair of interaction determination compositions 1404a, 1404 b can comprise different sequences.

Referring to FIG. 14B, the method can include ligating the interactiondetermination oligonucleotides 1412 a, 1412 b of the first pair ofinteraction determination compositions 1404 a, 1404 b using a bridgeoligonucleotide 1432 to generate a ligated interaction determinationoligonucleotide 1412. The bridge oligonucleotide 1432 can comprise twohybridization regions capable of specifically binding to the bridgeoligonucleotide hybridization regions 1428 a, 1428 b of the first pairof interaction determination compositions 1404 a, 1404 b.

Referring to FIG. 14C, the method can include barcoding the ligatedinteraction determination oligonucleotide 1412 using a plurality ofbarcodes 1440 to create a plurality of barcoded interactiondetermination oligonucleotides 1456. Each of the plurality of barcodes1440 can comprise a barcode sequence 1448 and a capture sequence 1444.The method can include obtaining sequencing data of the plurality ofbarcoded interaction determination oligonucleotides 1456. The method caninclude determining an interaction between the first and second cellularcomponent targets 1436 a, 1436 b based on the association of theinteraction determination sequences 1424 a, 1424 b of the first pair ofinteraction determination compositions 1404 a, 1404 b in the obtainedsequencing data. In some embodiments, at least one of the two cellularcomponent binding reagent can comprise a protein binding reagent. Theprotein binding reagent can be associated with one of the twointeraction determination oligonucleotides. The one or more cellularcomponent targets can comprise at least one protein target.

In some embodiments, contacting the cell with the first pair ofinteraction determination compositions 1404 a, 1404 b comprises:contacting the cell with each of the first pair of interactiondetermination compositions 1404 a, 1404 b sequentially orsimultaneously. The first protein target 1436 a can be the same as thesecond protein target 1436 b. The first protein target 1436 a can bedifferent from the second protein target 1436 b. The first and thesecond cellular component targets can be the same or different.

The interaction determination oligonucleotides 1412 a, 1412 be can havedifferent lengths in different implementations. In some embodiments, aninteraction determination oligonucleotide 1412 a, 1412 b is, or isabout, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860,870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000,or a number or a range between any two of these values, nucleotides inlength. In some embodiments, an interaction determinationoligonucleotide 1412 a, 1412 b is at least, or is at most, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340,350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760,770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000, nucleotides inlength.

In some embodiments, the method comprises: contacting the cell with asecond pair of interaction determination compositions. The cell cancomprise a third cellular component target and a fourth cellularcomponent target (e.g., a third protein target and a fourth proteintarget). Each of the second pair of interaction determinationcompositions can comprise a cellular component binding reagentassociated with an interaction determination oligonucleotide. Thecellular component binding reagent of one of the second pair ofinteraction determination compositions can be capable of specificallybinding to the third cellular component target and the cellularcomponent binding reagent of the other of the second pair of interactiondetermination compositions can be capable of specifically binding to thefourth cellular component target. In some embodiments, at least one ofthe third and fourth cellular component targets can be different fromone of the first and second cellular component targets. In someembodiments, at least one of the third and fourth cellular componenttargets and at least one of the first and second cellular componenttargets can be identical.

In some embodiments, the method comprises: contacting the cell withthree or more pairs of interaction determination compositions. Theinteraction determination sequences of a number of interactiondetermination compositions of the plurality of pairs of interactiondetermination compositions can comprise the same or different sequences.In some embodiments, the number of interaction determinationcompositions having interaction determination sequences with the same ordifferent sequences can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or anumber or a range between any two of these values. In some embodiments,the number of interaction determination compositions having interactiondetermination sequences with the same or different sequences can be atleast, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000.

In some embodiments, the bridge oligonucleotide hybridization regions1428 a, 1428 b of the first pair of interaction determinationcompositions 1404 a, 1404 b comprise different sequences. At least oneof the bridge oligonucleotide hybridization regions 1428 a, 1428 b canbe complementary to at least one of the two hybridization regions of thebridge oligonucleotide 1432.

In some embodiments, ligating the interaction determinationoligonucleotides 1412 a, 1412 b of the first pair of interactiondetermination compositions 1404 a, 1404 b using the bridgeoligonucleotide 1432 comprises: hybridizing a first hybridizationregions of the bridge oligonucleotide 1432 with a first bridgeoligonucleotide hybridization region 1428 a of the bridgeoligonucleotide hybridization regions of the interaction determinationoligonucleotides; hybridizing a second hybridization region of thebridge oligonucleotide with a second bridge oligonucleotidehybridization region 1428 b of the bridge oligonucleotide hybridizationregions of the interaction determination oligonucleotides; and ligatingthe interaction determination oligonucleotides 1428 a, 1428 b that arehybridized to the bridge oligonucleotide 1432 to generate a ligatedinteraction determination oligonucleotide.

In some embodiments, the at least one of the one or more cellularcomponent targets (e.g., protein targets 1436 a, 1436 b) is on a cellsurface. In some embodiments, the method can comprise: fixating the cellprior to contacting the cell with the first pair of interactiondetermination compositions 1404 a, 1404 b. In some embodiments, themethod can comprise: removing unbound interaction determinationcompositions of the first pair of interaction determination compositions1404 a, 1404 b. Removing the unbound interaction determinationcompositions can comprise washing the cell with a washing buffer.Removing the unbound interaction determination compositions can compriseselecting the cell using flow cytometry. In some embodiments, the methodcan comprise: lysing the cell.

In some embodiments, the interaction determination oligonucleotide isconfigured to be detachable or non-detachable from the protein bindingreagent. The method can comprise: detaching the interactiondetermination oligonucleotide 1412 a, 1412 b or the ligated interactiondetermination oligonucleotide 1412 from the cellular component bindingreagent. Detaching the interaction determination oligonucleotide 1412 a,1412 b can comprise detaching the interaction determinationoligonucleotide 1412 a, 1412 b from the cellular component bindingreagent by UV photocleaving, chemical treatment, heating, enzymetreatment, or any combination thereof. The interaction determinationoligonucleotide 1412 a, 1412 b may be not homologous to genomicsequences of the cell. The interaction determination oligonucleotide1412 a, 1412 b can be homologous to genomic sequences of a species.

In some embodiments, the interaction determination oligonucleotide 1412b of the one of the first pair of interaction determination compositions1404 a, 1404 b comprises a sequence 1420 complementary to the capturesequence 1444. The capture sequence 1444 can comprise a poly(dT) region.The sequence 1420 of the interaction determination oligonucleotidecomplementary to the capture sequence 1444 can comprise a poly(dA)region. In some embodiments, the interaction determinationoligonucleotide 1412 a, 1412 b comprises a second barcode sequence. Theinteraction determination oligonucleotide 1412 a of the other of thefirst pair of interaction identification compositions 1404 a, 1404 b cancomprise a binding site for a universal primer 1416. The interactiondetermination oligonucleotide 1404 a, 1404 b can be associated with adetectable moiety.

In some embodiments, the cellular component binding reagent can beassociated with two or more interaction determination oligonucleotides1412 a, 1412 b with the same or different interaction determinationsequences 1428 a, 1428 b. In some embodiments, one of the plurality ofinteraction determination compositions 1404 a, 1404 b comprises a secondprotein binding reagent not associated with the interactiondetermination oligonucleotide. The cellular component binding reagentand the second cellular component binding reagent can be identical. Thecellular component binding reagent can be associated with a detectablemoiety. The cellular component binding reagent 1404 a, 1404 b can beassociated with two or more interaction determination oligonucleotides1412 a, 1412 b with an identical sequence.

In some embodiments, the method comprises: contacting two or more cellswith the first pair of interaction determination compositions 1404 a,1404 b. Each of the two or more cells can comprise the first and thesecond cellular component targets (e.g., the first and the secondprotein targets 1436 a, 1436 b). At least one of the two or more cellscan comprise a single cell.

Referring to FIG. 14B, the barcode 1440 comprises a cell label sequence1448, a binding site for a universal primer, or any combination thereof.At least two barcodes of the plurality of barcodes can comprise anidentical cell label sequence 1448.

In some embodiments, the cellular component target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. The cellular component target can be, or comprise, a lipid, acarbohydrate, or any combination thereof. The cellular component targetcan be selected from a group comprising 10-100 different cellularcomponent targets.

In some embodiments, the plurality of barcodes is associated with aparticle (e.g., a bead 1438). At least one barcode the plurality ofbarcodes can be immobilized on the particle. At least one barcode of theplurality of barcodes can be partially immobilized on the particle. Atleast one barcode of the plurality of barcodes can be enclosed in theparticle. At least one barcode of the plurality of barcodes can bepartially enclosed in the particle. The particle can be disruptable. Insome embodiments, the barcodes of the particle comprise barcodesequences selected from at least 1000, 10000, or more different barcodesequences

Referring to FIG. 14C, barcoding the interaction determinationoligonucleotides 1412 using the plurality of barcodes 1440 comprises:contacting the plurality of barcodes 1440 with the interactiondetermination oligonucleotides 1412 to generate barcodes hybridized tothe interaction determination oligonucleotides; and extending thebarcodes hybridized to the interaction determination oligonucleotides togenerate the plurality of barcoded interaction determinationoligonucleotides 1456. Extending the barcodes 1440 can compriseextending the barcodes 1440 using a DNA polymerase to generate theplurality of barcoded interaction determination oligonucleotides 1456.Extending the barcodes 1440 can comprise extending the barcodes 1440using a reverse transcriptase to generate the plurality of barcodedinteraction determination oligonucleotides 14. Extending the barcodes 14can comprise extending the barcodes 14 using a Moloney Murine LeukemiaVirus (M-MLV) reverse transcriptase or a Taq DNA polymerase to generatethe plurality of barcoded interaction determination oligonucleotides1456. Extending the barcodes 14 can comprise displacing the bridgeoligonucleotide 1432 from the ligated interaction determinationoligonucleotide 1412.

Referring to FIGS. 14D and 14E, the method can comprise: amplifying theplurality of barcoded interaction determination oligonucleotides 1456 toproduce a plurality of amplicons. Amplifying the plurality of barcodedinteraction determination oligonucleotides 1456 can comprise amplifying,using polymerase chain reaction (PCR), at least a portion of the barcodesequence 1448 and at least a portion of the interaction determinationoligonucleotide 1428 a, 1428 b. Obtaining the sequencing data of theplurality of barcoded interaction determination oligonucleotides 1456can comprise obtaining sequencing data of the plurality of amplicons.Obtaining the sequencing data can comprise sequencing at least a portionof the barcode sequence 1448 and at least a portion of the interactiondetermination oligonucleotide 1428 a, 1428 b. In some embodiments,obtaining sequencing data of the plurality of barcoded interactiondetermination oligonucleotides 1456 comprises obtaining partial and/orcomplete sequences of the plurality of barcoded interactiondetermination oligonucleotides 1456 (or a reverse, a complement, areverse complement 1456 r, or any combination thereof).

Referring to FIG. 14C, the plurality of barcodes 1440 can comprise aplurality of stochastic barcodes. The barcode sequence of each of theplurality of stochastic barcodes can comprise a molecular label sequence1448. The molecular label sequences 1448 of at least two stochasticbarcodes of the plurality of stochastic barcodes can comprise differentsequences. Barcoding the interaction determination oligonucleotides 1412using the plurality of barcodes 1440 to create the plurality of barcodedinteraction determination oligonucleotides 1456 can comprisestochastically barcoding the interaction determination oligonucleotides1412 using the plurality of stochastic barcodes to create a plurality ofstochastically barcoded interaction determination oligonucleotides.

The method can also be used with other single cell RNA sequencingmethods. For example, one single workflow can be used to determine mRNAexpression levels, protein expression levels (or expression levels ofcellular component targets), and/or protein-protein interactions (orinteractions between cellular components). As a result, in a singleexperiment, data about mRNA expression, protein expression, andprotein-protein interactions from in a single cell (or multiple singlecells) can be obtained. In some embodiments, the method comprises:barcoding a plurality of targets (e.g., mRNA species of interest) of thecell using the plurality of barcodes 1440 to create a plurality ofbarcoded targets; and obtaining sequencing data of the barcoded targets.Barcoding the plurality of targets using the plurality of barcodes 1440to create the plurality of barcoded targets can comprise: contactingcopies of the targets with target-binding regions (e.g., the poly(dT)region 1444) of the barcodes 1440; and reverse transcribing theplurality targets using the plurality of barcodes 1440 to create aplurality of reverse transcribed targets. The method can comprise: priorto obtaining the sequencing data of the plurality of barcoded targets,amplifying the barcoded targets to create a plurality of amplifiedbarcoded targets (similar to amplifying the barcoded interactiondetermination oligonucleotides 1456 illustrated in FIGS. 14E and 14F).Amplifying the barcoded targets to generate the plurality of amplifiedbarcoded targets can comprise: amplifying the barcoded targets bypolymerase chain reaction (PCR). Barcoding the plurality of targets ofthe cell using the plurality of barcodes 1440 to create the plurality ofbarcoded targets can comprise stochastically barcoding the plurality oftargets of the cell using the plurality of stochastic barcodes to createa plurality of stochastically barcoded targets.

Embodiments disclosed herein also include kits for identifying cellularcomponent-cellular component interactions (e.g., protein-proteininteractions). In some embodiments, the kit comprises: a first pair ofinteraction determination compositions, wherein each of the first pairof interaction determination compositions comprises a cellular componentbinding reagent associated with an interaction determinationoligonucleotide, wherein the cellular component binding reagent of oneof the first pair of interaction determination compositions is capableof specifically binding to a first cellular component target and acellular component binding reagent of the other of the first pair ofinteraction determination compositions is capable of specificallybinding to the second cellular component target, wherein the interactiondetermination oligonucleotide comprises an interaction determinationsequence and a bridge oligonucleotide hybridization region, and whereinthe interaction determination sequences of the first pair of interactiondetermination compositions comprise different sequences; and a pluralityof bridge oligonucleotides each comprising two hybridization regionscapable of specifically binding to the bridge oligonucleotidehybridization regions of the first pair of interaction determinationcompositions.

In some embodiments, the kit comprises: a second pair of interactiondetermination compositions, wherein each of the second pair ofinteraction determination compositions comprises a cellular componentbinding reagent associated with an interaction determinationoligonucleotide, wherein the cellular component binding reagent of oneof the second pair of interaction determination compositions is capableof specifically binding to a third cellular component target and thecellular component binding reagent of the other of the second pair ofinteraction determination compositions is capable of specificallybinding to a fourth cellular component target. In some embodiments, thekit comprises: three or more pairs of interaction determinationcompositions.

In some embodiments, the kit comprises: a plurality of barcodes, whereineach of the plurality of barcodes comprises a barcode sequence and acapture sequence. The plurality of barcodes can comprise a plurality ofstochastic barcodes, wherein the barcode sequence of each of theplurality of stochastic barcodes comprises a barcode sequence (e.g., amolecular label sequence), wherein the barcode sequences of at least twostochastic barcodes of the plurality of stochastic barcodes comprisedifferent sequences. In some embodiments, the plurality of barcodes isassociated with a particle. At least one barcode of the plurality ofbarcodes can be immobilized on the particle, partially immobilized onthe particle, enclosed in the particle, partially enclosed in theparticle, or any combination thereof. The particle can be disruptable.The particle can comprise a bead.

In some embodiments, the kit comprises: a DNA polymerase. The kit cancomprise a reverse transcriptase. The kit can comprise: a Moloney MurineLeukemia Virus (M-MLV) reverse transcriptase or a Taq DNA polymerase. Insome embodiments, the method comprises a fixation agent (e.g., formalin,paraformaldehyde, glutaraldehyde/osmium tetroxide, Alcoholic fixatives,Hepes-glutamic acid buffer-mediated organic solvent protection effect(HOPE), Bouin solution, or any combination thereof).

EXAMPLES

Some aspects of the embodiments discussed above are disclosed in furtherdetail in the following examples, which are not in any way intended tolimit the scope of the present disclosure.

Example 1 Oligonucleotides for Conjugation with Protein Binding Reagents

This example demonstrates designing of oligonucleotides that can beconjugated with protein binding reagents. The oligonucleotides can beused to determine protein expression and gene expression simultaneously.The oligonucleotides can also be used for sample indexing to determinecells of the same or different samples.

95mer Oligonucleotide Design

The following method was used to generate candidate oligonucleotidesequences and corresponding primer sequences for simultaneousdetermination of protein expression and gene expression or sampleindexing.

1. Sequence Generation and Elimination

The following process was used to generate candidate oligonucleotidesequences for simultaneous determination of protein expression and geneexpression or sample indexing.

Step 1a. Randomly generate a number of candidate sequences (50000sequences) with the desired length (45 bps).

Step 1b. Append the transcriptional regulator LSRR sequence to the 5′end of the sequences generated and a poly(A) sequence (25 bps) to the 3′end of the sequences generated.

Step 1c. Remove sequences generated and appended that do not have GCcontents in the range of 40% to 50%.

Step 1d. Remove remaining sequences with one or more hairpin structureseach.

The number of remaining candidate oligonucleotide sequences was 423.

2. Primer Design

The following method was used to design primers for the remaining 423candidate oligonucleotide sequences.

2.1 N1 Primer: Use the universal N1 sequence:5′-GTTGTCAAGATGCTACCGTTCAGAG-3′ (LSRR sequence; SEQ ID NO. 5) as the N1primer.

2.2 N2 Primer (for amplifying specific sample index oligonucleotides;e.g., N2 primer in FIGS. 15B-15D):

2.2a. Remove candidate N2 primers that do not start downstream of the N1sequence.

2.2b. Remove candidate N2 primers that overlap in the last 35 bps of thecandidate oligonucleotide sequence.

2.2c. Remove the primer candidates that are aligned to the transcriptomeof the species of cells being studied using the oligonucleotides (e.g.,the human transcriptome or the mouse transcriptome).

2.2d. Use the ILR2 sequence as the default control(ACACGACGCTCTTCCGATCT; SEQ ID NO. 8) to minimize or avoid primer-primerinteractions.

Of the 423 candidate oligonucleotide sequences, N2 primers for 390candidates were designed.

3. Filtering

The following process was used to filter the remaining 390 candidateprimer sequences.

3a. Eliminate any candidate oligonucleotide sequence with a randomsequence ending in As (i.e., the effective length of the poly(A)sequence is greater than 25 bps) to keep poly(A) tail the same lengthfor all barcodes.

3b. Eliminate any candidate oligonucleotide sequences with 4 or moreconsecutive Gs (>3Gs) because of extra cost and potentially lower yieldin oligo synthesis of runs of Gs.

FIG. 15A shows a non-limiting exemplary candidate oligonucleotidesequence generated using the method above.

200mer Oligonucleotide Design

The following method was used to generate candidate oligonucleotidesequences and corresponding primer sequences for simultaneousdetermination of protein expression and gene expression and sampleindexing.

1. Sequence Generation and Elimination

The following was used to generate candidate oligonucleotide sequencesfor simultaneous determination of protein expression and gene expressionand sample indexing.

1a. Randomly generate a number of candidate sequences (100000 sequences)with the desired length (128 bps).

1b. Append the transcriptional regulator LSRR sequence and an additionalanchor sequence that is non-human, non-mouse to the 5′ end of thesequences generated and a poly(A) sequence (25 bps) to the 3′ end of thesequences generated.

1c. Remove sequences generated and appended that do not have GC contentsin the range of 40% to 50%.

1d. Sort remaining candidate oligonucleotide sequences based on hairpinstructure scores.

1e. Select 1000 remaining candidate oligonucleotide sequences with thelowest hairpin scores.

2. Primer Design

The following method was used to design primers for 400 candidateoligonucleotide sequences with the lowest hairpin scores.

2.1 N1 Primer: Use the universal N1 sequence:5′-GTTGTCAAGATGCTACCGTTCAGAG-3′ (LSRR sequence; SEQ ID NO. 5) as the N1primer.

2.2 N2 Primer (for amplifying specific sample index oligonucleotides;e.g., N2 primer in FIGS. 15B and 15C):

2.2a. Remove candidate N2 primers that do not start 23 nts downstream ofthe N1 sequence (The anchor sequence was universal across all candidateoligonucleotide sequences).

2.2b. Remove candidate N2 primers that overlap in the last 100 bps ofthe target sequence. The resulting primer candidates can be between the48th nucleotide and 100th nucleotide of the target sequence.

2.2c. Remove the primer candidates that are aligned to the transcriptomeof the species of cells being studied using the oligonucleotides (e.g.,the human transcriptome or the mouse transcriptome).

2.2d. Use the ILR2 sequence, 5′-ACACGACGCTCTTCCGATCT-3′ (SEQ ID NO. 8)as the default control to minimize or avoid primer-primer interactions.

2.2e. Remove N2 primer candidates that overlap in the last 100 bps ofthe target sequence.

Of the 400 candidate oligonucleotide sequences, N2 primers for 392candidates were designed.

3. Filtering

The following was used to filter the remaining 392 candidate primersequences.

3a. Eliminate any candidate oligonucleotide sequence with a randomsequence ending in As (i.e., the effective length of the poly(A)sequence is greater than 25 bps) to keep poly(A) tail the same lengthfor all barcodes.

3b. Eliminate any candidate oligonucleotide sequences with 4 or moreconsecutive Gs (>3Gs) because of extra cost and potentially lower yieldin oligo synthesis of runs of Gs.

FIG. 15B shows a non-limiting exemplary candidate oligonucleotidesequence generated using the method above. The nested N2 primer shown inFIG. 15B can bind to the antibody or sample specific sequence fortargeted amplification. FIG. 15C shows the same non-limiting exemplarycandidate oligonucleotide sequence with a nested universal N2 primerthat corresponds to the anchor sequence for targeted amplification. FIG.15D shows the same non-limiting exemplary candidate oligonucleotidesequence with a N2 primer for one step targeted amplification.

Altogether, these data indicate that oligonucleotide sequences ofdifferent lengths can be designed for simultaneous determination ofprotein expression and gene expression or sample indexing. Theoligonucleotide sequences can include a universal primer sequence, anantibody specific oligonucleotide sequence or a sample indexingsequence, and a poly(A) sequence.

Example 2 Comparison of Detection Sensitivity with Different Antibody:Oligonucleotide Ratios

This example demonstrates detection sensitivity of CD4 protein using ananti-CD4 antibody conjugated with 1, 2, or 3 oligonucleotides.

Frozen peripheral blood mononuclear cells (PBMCs) of a subject werethawed. The thawed PBMCs were stained with three types of anti-CD4antibody at 0.06 μg/100 μl (1:333 dilution of oligonucleotide-conjugatedantibody stocks) at room temperature for 20 minutes. Each type of thetypes of anti-CD4 antibody was conjugated with 1, 2, or 3oligonucleotides (“antibody oligonucleotides”). The sequence of theantibody oligonucleotide is shown in FIG. 16. The cells were washed toremove unbound anti-CD4 antibodies. The cells were stained with CalceinAM (BD (Franklin Lake, N.J.)) and Drag7™ (Abcam (Cambridge, UnitedKingdom)) for sorting with flow cytometry to obtain live cells. Thecells were washed to remove excess Calcein AM and Drag7™. Single cellsstained with Calcein AM (live cells) and not Drag7™ (cells that were notdead or permeabilized) were sorted, using flow cytometry, into a BDRhapsody™ cartridge.

Of the wells containing a single cell and a bead, 3500 of the singlecells in the wells were lysed in a lysis buffer with 5 mM DTT. The CD4mRNA expression profile was determined using BD Rhapsody™ beads. The CD4protein expression profile was determined using BD Rhapsody™ beads andthe antibody oligonucleotides. Briefly, the mRNA molecules were releasedafter cell lysis. The Rhapsody™ beads were associated with stochasticbarcodes each containing a molecular label, a cell label, and a polyTregion. The poly(A) regions of the mRNA molecules released from thelysed cells hybridized to the polyT regions of the stochastic barcodes.The poly(A) regions of the oligonucleotides hybridized to the polyTregions of the stochastic barcodes. The mRNA molecules were reversetranscribed using the stochastic barcodes. The antibody oligonucleotideswere replicated using the stochastic barcodes. The reverse transcriptionand replication occurred in one sample aliquot at the same time.

The reverse transcribed products and replicated products were PCRamplified for 15 cycles at 60 degrees annealing temperature usingprimers for determining the mRNA expression profiles of 488 blood panelgenes, using blood panel N1 primers, and the expression profile of CD4protein, using the antibody oligonucleotide N1 primers (“PCR 1”). Excessstochastic barcodes were removed with Ampure cleanup. The products fromPCR1 were divided into two aliquots, one aliquot for determining themRNA expression profiles of the 488 blood panel genes, using the bloodpanel N2 primers, and one aliquot for determining the expression profileof CD4 protein, using the antibody oligonucleotide N2 primers (“PCR 2”).Both aliquots were PCR amplified for 15 cycles at 60 degrees annealingtemperature. The expression of CD4 protein in the lysed cells wasdetermined based on the antibody oligonucleotides as illustrated in FIG.16 (“PCR 2”). Sequencing data was obtained and analyzed after sequencingadaptor ligation (“PCR 3”). Cell types were determined based on theexpression profiles of the 488 blood panel genes.

FIGS. 17A-17F are non-limiting exemplary t-Distributed StochasticNeighbor Embedding (tSNE) projection plots showing results of usingoligonucleotide-conjugated antibodies to measure CD4 protein expressionand gene expression simultaneously in a high throughput manner. CD4protein expression was distinctly and robustly detected in CD4expressing cell types (e.g., CD4 T cells) with anti-CD4 antibodiesconjugated to 1, 2, or 3 antibody oligonucleotides (FIGS. 17B, 17D, and17F respectively). FIGS. 18A-18F are non-limiting exemplary bar chartsshowing the expressions of CD4 mRNA and protein in CD4 T cells (high CD4expression), CD8 T cells (minimal CD4 expression), and Myeloid cells(some CD4 expression). With similar sequencing depth, detectionsensitivity for CD4 protein level increased with higher ratios ofantibody:oligonucleotide, with the 1:3 ratio performing better than the1:1 and 1:2 ratios (FIG. 19). The expression of CD4 protein on cellsurface of cells sorted using flow cytometry was confirmed using FlowJo(FlowJo (Ashland, Oreg.)) as shown in FIGS. 20A-20D. FIGS. 21A-21F arenon-limiting exemplary bar charts showing the expressions of CD4 mRNAand CD4 protein in CD4 T cells, CD8 T cells, and Myeloid cells of twosamples. The second sample was prepared using two different samplepreparation protocols. FIG. 22 is a non-limiting exemplary bar chartshowing detection sensitivity for CD4 protein level determined usingdifferent sample preparation protocols with an antibody:oligonucleotideratio of 1:3.

Altogether, these data indicate that CD4 protein expression can bedistinctly and robustly detected based on oligonucleotide-conjugatedwith anti-CD4 antibodies. Detection sensitivity for CD4 protein levelcan increase with higher antibody:oligonucleotide ratios.

Example 3 Sample Indexing

This example demonstrates identifying cells of different samples usingsample indexing with high labeling efficiency and low non-specificlabeling or spill over. This example also demonstrates the effects ofthe lengths of the sample indexing oligonucleotides and the cleavabilityof sample indexing oligonucleotides on sample indexing.

FIG. 23 shows a non-limiting exemplary experimental design forperforming sample indexing and determining the effects of the lengths ofthe sample indexing oligonucleotides and the cleavability of sampleindexing oligonucleotides on sample indexing. The left column in FIG. 23shows that Jurkat cells were labeled with anti-CD147 antibodies withdifferent sample indexing oligonucleotides to determine the effects ofthe lengths of the sample indexing oligonucleotides and the cleavabilityof sample indexing oligonucleotides on sample indexing. Theoligonucleotide-conjugated with the anti-CD147 antibodies were 200nucleotides in length that were cleavable from the antibody (T-linker),95 nucleotides in length that were cleavable (T-linker), and 95nucleotides in length that were not cleavable. After further samplepreparation, the effects of the lengths of the sample indexingoligonucleotides and the cleavability of sample indexingoligonucleotides on sample indexing were determined using a Rhapsody™flowcell (labeled “Flowcell 1” in FIG. 23, abbreviated as “FC1”). Samplepreparation included Calcein AM and Draq7™ staining, cell sorting usingflow cytometry, and stochastic barcoding.

The middle column in FIG. 23 shows that cells were labeled withanti-CD147 antibodies conjugated with three different sample indexingoligonucleotides that were cleavable (the sample indexingoligonucleotides, labeled “Short 1,” “Short 2,” and “Short 3” in FIG.23, can be abbreviated as S1, S2, and S3). Ramos cells were labeled withtwo types of anti-CD147 antibodies conjugated with different sampleindexing oligonucleotides that are cleavable (the sample indexingoligonucleotides are labeled “Short 1” and “Short 2” in FIG. 23). Jurkatcells were labeled with two types of anti-CD147 antibodies conjugatedwith different sample indexing oligonucleotides that are cleavable (thesample indexing oligonucleotides are labeled “Short 1” and “Short 3” inFIG. 23). The labeled cells were mixed in a 1:1 ratio, followed byfurther sample preparation and determination of the efficiency andspecificity of sample indexing using a Rhapsody™ flowcell (labeled“Flowcell 2” in FIG. 23, abbreviated as “FC2”). The right column in FIG.23 shows the control experiment where Jurkat cells and Ramos cells thatwere not labeled were mixed in a 1:1 ratio prior to further samplepreparation and analysis using a Rhapsody™ flowcell (labeled as“Flowcell 3” in FIG. 23, abbreviated as “FC3”).

The cells were stained with antibodies conjugated with sample indexingoligonucleotides a 1:3 antibody:oligonucleotide ratio at roomtemperature for 20 minutes. The antibodies were diluted fromoligonucleotide-conjugated antibody stocks at 1:20 dilution in 100 μlusing a pH 7.5 diluent. The cells were washed to remove unboundanti-CD147 antibodies. The cells were stained with Calcein AM (stainingfor live cells) and Drag7™ (staining for cells that were dead orpermeabilized)) for sorting with flow cytometry to obtain live cells.The cells were washed to remove excess Calcein AM and Drag7™. Singlecells stained with Calcein AM and not Drag7™ were sorted, using flowcytometry, into a BD Rhapsody™ cartridge containing a flowcell.

Of the wells containing a single cell and a bead, 1000 of the singlecells in the wells were lysed in a lysis buffer. The CD147 mRNAexpression profile was determined using BD Rhapsody™ beads. Briefly, themRNA molecules were released after cell lysis. The Rhapsody™ beads wereassociated with stochastic barcodes each containing a molecular label, acell label, and a polyT region. The poly(A) regions of the mRNAmolecules released from the lysed cells hybridized to the polyT regionsof the stochastic barcodes. The poly(A) regions of the sample indexingoligonucleotides, cleaved from the antibodies if cleavable, hybridizedto the polyT regions of the stochastic barcodes. The mRNA molecules werereverse transcribed using the stochastic barcodes. The antibodyoligonucleotides were replicated using the stochastic barcodes. Thereverse transcription and replication occurred in one sample aliquot atthe same time for 15 cycles at 60 degrees annealing temperature.

The reverse transcribed products and replicated products were PCRamplified for 15 cycles at 60 degrees annealing temperature usingprimers for determining the mRNA expression profiles of 488 blood panelgenes, using blood panel N1 primers, and the expression of CD147protein, using the sample indexing oligonucleotide N1 primers (“PCR 1”).Excess primers were removed with Ampure cleanup. The products from PCR1were further PCR amplified (“PCR 2”) for 15 cycles at 60 degreesannealing temperature using blood panel N2 primers and sample indexingoligonucleotide N1 primers with a flanking sequence for adaptorligation. Sequencing data was obtained and analyzed after sequencingadaptor ligation (“PCR 3”). Cell types were determined based on theexpression profiles of the 488 blood panel genes.

Table 1 shows metrics of the sequencing data obtained using theexperimental design illustrated in FIG. 23. Three types of anti-CD147antibody, conjugated with any one of the three types of sample indexingoligonucleotides (cleavable 95mer, non-cleavable 95mer, and cleavable200mer shown at the left column in FIG. 23), were successfully used forsample indexing. More than 11% of total numbers of reads in thesequencing data were attributed to the sample indexing oligonucleotides.

TABLE 1 Sequencing metrics of sample indexing oligonucleotides. FC1(Oligos) FC2 (sample indexing) Sample Jurkat (J) Ramos 95mer, 95mer,non- 200mer, J & R (R) J Oligo type cleavable cleavable cleavable 95mer,cleavable % of total reads 11.4 22.4 11.8 16.7 12.9 11 % of totalmolecules 29 41.4 7.5 28.1 20.3 16.3 Average # of mols per cell 10431679 303 NA NA NA Median # of mols per cell 895 1536 256 NA NA NA OligoRSEC seq depth 3.9 4.6 11.54 1.74 1.87 2 Blood panel RSEC seq depth10.72 10.72 10.72 4.1 4.1 4.1

Three types of anti-CD147 antibody, conjugated with any one of the threecleavable 95mers (labeled “Short 1,” “Short 2,” and “Short 3” at themiddle column in FIG. 23), were successfully used for sample indexing todistinguish Ramos and Jurkat cells of different samples. More than 11%of the total number of reads in the sequencing data were attributed tothe sample indexing oligonucleotides. The percentage was the highest(16.7%) for a sample containing both Jurkat and Ramos cells.

FIGS. 24A-24C are non-limiting exemplary tSNE plots of expressionprofiles of cells showing that the three types of anti-CD147 antibodyconjugated with different sample indexing oligonucleotides (cleavable95mer, non-cleavable 95mer, and cleavable 200mer) can be used fordetermining the protein expression level of CD147. The overlays of theCD147 expression, determined using sample indexing oligonucleotides, ontSNE projection plots of the expression profiles of the cells show thatthe CD147 expression patterns determined using different sample indexingoligonucleotides were similar. Detection of the three sample indexingoligonucleotides was 100%. FIG. 25 is a non-limiting exemplary tSNE plotwith an overlay of GAPDH expression per cell. “Cells” at the top rightcorner of the plot were low in GAPDH, suggesting that they were not realcells. These “cells” were high in sample indexing oligonucleotides.

FIGS. 26A-26C are non-limiting exemplary histograms showing the numberof molecules of sample indexing oligonucleotides detected using thethree types of sample indexing oligonucleotides. The non-cleavablesample indexing oligonucleotide was the most sensitive in terms of thenumber of molecules detected.

FIGS. 27A-27C are plots and bar charts showing that CD147 expression washigher in dividing cells (determined using “Flowcell 1” in FIG. 23).CD147 expression was not uniform across cells in the sample of Jurkatcells. Cells can be classified based on the cell cycle gene AURKB. ThemRNA expression of the AURKB gene correlated with the CD147 proteinexpression determined using sample indexing. FIG. 27C shows a comparisonof CD147 expression, determined using the three sample indexingoligonucleotides, in AURKB+ and AURKB− cells.

FIGS. 28A-28C are non-limiting exemplary tSNE projection plots ofexpression profiles of cells, showing that sample indexing can be usedto identify cells of different samples. Reads of sample indexingoligonucleotides in the sequencing data, containing expression profilesof the 488 blood panel genes, were filtered. The tSNE projection plotsin FIGS. 28A-28C were generated using the filtered sequencing data. Thecluster corresponding to Jurkat cells and the cluster corresponding toRamos cells (determined based on the abundance of the sample indexingoligonucleotides labeled “Short 1” in FIG. 23) are clearly separated inthe tSNE projection plot of FIG. 28A (similarly for FIGS. 28B-28C).

Sample indexing correctly matched the cell type. For example, FIG. 28Bshows that Ramos cells, which were labeled with the sample indexingoligonucleotides labeled “Short 2” in FIG. 23, had high expression ofCD147, while Jurkat cells which were not labeled had minimal expressionof CD147. FIG. 28C shows that Jurkat cells, which were labeled with thesample indexing oligonucleotides labeled “Short 3” in FIG. 23, had highexpression of CD147, while Ramos cells which were not labeled had lowexpression of CD147. The small doublet cluster at 3.6% frequency closelymatched the expected doublet rate of 3.92% from image analysis ofoccupancy of wells.

FIGS. 29A-29C are non-limiting exemplary histograms of the sampleindexing sequences per cell based on the numbers of molecules of thesample indexing oligonucleotides determined. The sequencing data wascorrected with distribution based error correction (DBEC) with a cutoffof 250. DBEC has been described in U.S. patent application Ser. No.15/605,874, filed on May 25, 2017, the content of which is incorporatedherein by reference in its entirety. After DBEC, there seemed to be aclear “noise” peak in the 100s that was distinguishable from the“signal” peak in the 1000s (FIGS. 29B-29C). FIGS. 29B-29C also show thatthe detection of sample indexing oligonucleotides to be in the high 100safter a noise subtraction of 250.

FIGS. 30A-30D are non-limiting exemplary plots comparing annotations ofcell types determined based on mRNA expression of CD3D (for Jurkatcells) and JCHAIN (for Ramos cells) and sample indexing of Jurkat andRamos cells. FIG. 30A is a non-limiting exemplary histogram showingclear separation of a signal peak of JCHAIN expression and noise signalswith low numbers of molecules of sample indexing oligoncleotides percell. FIG. 30B is a non-limiting exemplary histogram showing clearseparation of a signal peak of Ramos expression and noise signals withlow numbers of molecules of sample indexing oligonucleotides per cell.Doublets were identified as cells with more than one sample indexingsequence with more than 250 counts. In FIG. 30C, Ramos cells wereidentified using the mRNA expression of JCHAIN, and Jurkat cells wereidentified using the mRNA expression of CD3D.

Ramos cells were labeled with two types of anti-CD147 antibodiesconjugated with different sample indexing oligonucleotides that arecleavable (the sample indexing oligonucleotides are labeled “Short 1”and “Short 2” in FIG. 23). Jurkat cells were labeled with two types ofanti-CD147 antibodies conjugated with different sample indexingoligonucleotides that are cleavable (the sample indexingoligonucleotides are labeled “Short 1” and “Short 3” in FIG. 23). InFIG. 30D, the identity of the Ramos cells were identified based on thenumbers of counts of the “Short 2” sample indexing oligonucleotide(greater than 250) in the sequencing data, and the identity of theJurkat cells were identified based on the numbers of counts of the“Short 3” sample indexing oligonucleotide (greater than 250) in thesequencing data.

FIGS. 31A-31C are non-limiting tSNE projection plots of the mRNAexpression profiles of Jurkat and Ramos cells with overlays of the mRNAexpressions of CD3D and JCHAIN (FIG. 31A), JCHAIN (FIG. 31B), and CD3D(FIG. 31C). The mRNA expression of JCHAIN was higher in Ramos cells(FIG. 31B) and minimal in Jurkat cells (FIG. 31C) as expected. The mRNAexpression of CD3D was higher in Jurkat cells (FIG. 31C) and minimal inRamos cells (FIG. 31B).

The comparisons of FIGS. 28B-28C with FIGS. 31B-31C revealed the highperformance of sample indexing in distinguishing cells of differentsamples. The performance of sample indexing in determining sample originis shown in FIG. 32. FIG. 32 is a non-limiting exemplary tSNE projectionplot of expression profiles of Jurkat and Ramos cells with an overlay ofthe cell types determined using sample indexing with a DBEC cutoff of250. Table 2 is a summary of the cell types determined using sampleindexing shown in FIG. 32. Table 3 shows the sensitivity and specificityof sample indexing, with doublets and undefined cells in Table 2excluded, in determining sample origin.

TABLE 2 Summary of sample origin determination using sample indexing. S2S2 + S3 S3 not labeled Doublets 0 36 1 0 Jurkat 0 5 658 12 Ramos 833 4 00 Unknown 30 0 17 0

TABLE 3 Specificity and sensitivity of sample indexing in determiningsample origin. Specificity Sensitivity S2 (Ramos)  99.5% (833/(833 + 4)) 100% (833/(833 + 0)) S3 (Jurkat) 99.25% (658/(658 + 4)) 98.2%(658/(658 + 12))

FIGS. 33A-33C are non-limiting exemplary bar charts of the numbers ofmolecules of sample indexing oligonucleotides per cell for Ramos &Jurkat cells (FIG. 33), Ramos cells (FIG. 33B), and Jurkat cells (FIG.33C) that were not labeled or labeled with “Short 3” sample indexingoligonucleotides, “Short 2” & “Short 3” sample indexingoligonucleotides, and “Short 2” sample indexing oligonucleotides. Lessthan 1% of single cells were labeled with both the “Short 2” and “Short3” sample indexing oligonucleotides FIGS. 34A-34C).

FIGS. 35A-35C are non-limiting exemplary tSNE plots showing batcheffects on expression profiles of Jurkat and Ramos cells between samplesprepared using different flowcells as outlined in FIG. 23. FIG. 35Ashows a non-limiting exemplary tSNE projection plot of expressionprofiles of Jurkat and Ramos cells with an overlay of different samplepreparations of the cells. The variations in mRNA expression profiles inthe 488 blood panel genes (FIG. 35B) and the CD3D gene (FIG. 35C)between different sample preparations may be a result of batch effectdue to different sequencing depths of the samples prepared using thedifferent flowcells.

Altogether, these data indicate that all three versions of sampleindexing oligonucleotides (95mer cleavable, 95mer non-cleavable, and200mer cleavable) can be used for sample indexing, with the 95mernon-cleavable having the highest efficiency. Sample indexing fordetermining sample origin can have high specificity and sensitivity.

Example 4 Hot:Cold Antibody Titration

This example demonstrates determining a ratio ofoligonucleotide-conjugated antibodies (“hot antibodies”) and antibodiesnot conjugated with oligonucleotides (“cold antibody”) such that theantibody oligonucleotides account for a desired percentage (e.g., 2%) oftotal reads in sequencing data.

An anti-CD147 antibody stock was diluted at 1:20, 1:100, 1:200, 1:300,1:400, 1:600, 1:800, and 1:1000 dilutions with PE buffer. Around 150000Jurkat cells in 100 μl of staining buffer (FBS) (BD (Franklin Lake,N.J.)) were stained at various antibody dilutions for 20 minutes at roomtemperature. After staining, the cells were washed once with 500 μl ofstaining buffer and resuspended in 200 μl for measurement offluorescence intensity. Muse™ Autophagy LC3-antibody (EMD Millipore(Billerica, Mass.)) was used to detect the anti-CD147 antibody bound tothe Jurkat cells. The fluorescence intensities from cells stained atvarious anti-CD147 antibody dilutions or cells not stained weredetermined and compared to determine an optimal dilution for theantibody (FIGS. 36A1-36A9, 36B and 36C). Fluorescence intensitydecreased with higher dilution. More than 99% of the cells were stainedwith a dilution ratio of 1:800. Fluorescence signals began to drop outat 1:800. Cells were stained to saturation up to a dilution ratio of1:200. Cells were stained close to saturation up to a dilution ratio of1:400.

FIG. 37 shows a non-limiting exemplary experimental design fordetermining a staining concentration of oligonucleotide-conjugatedantibodies such that the antibody oligonucleotides account for a desiredpercentage of total reads in sequencing data. An anti-CD147 antibody wasconjugated with a cleavable 95mer antibody oligonucleotide at anantibody:oligonucleotide ratio of 1:3 (“hot antibody”). The hot antibodywas diluted using a pH 7.5 diluent at a 1:100 ratio or a 1:800 ratio. Amixture of 10% hot antibody:90% cold antibody was prepared using 9 μl ofcold anti-CD147 antibody and 1 μl of the hot antibody. A mixture of 1%hot antibody:90% cold antibody was prepared using 9 μl of the coldanti-CD147 antibody and 1 μl of the mixture of 10% hot antibody:90% coldantibody.

Thawed peripheral blood mononuclear cells (PBMCs) with around 0.5million cells were stained in 100 μl of staining buffer (FBS) with the1:100 diluted stock with 100% hot antibody (1% of the stock hotantibody), the mixture of 10% hot antibody:90% cold antibody (0.1% ofthe stock hot antibody), the mixture of 1% hot antibody:99% coldantibody (0.01% of the stock hot antibody), and the 1:800 diluted stockwith 100% hot antibody (0.0125% of the stock hot antibody). Afterstaining, the cells were washed to remove unbound antibody molecules.The cells were stained with Calcein AM and Drag7™ for sorting with flowcytometry to obtain live cells. The cells were washed to remove excessCalcein AM and Drag7™. Single cells stained with Calcein AM (live cells)and not Drag7™ (cells that were not dead or permeabilized) were sorted,using flow cytometry, into a BD Rhapsody™ cartridge.

Of the wells containing a single cell and a bead, 1000 of the singlecells in the wells were lysed in a lysis buffer. For each single cell,the mRNA molecules were reverse transcribed and the antibodyoligonucleotides were replicated using stochastic barcodes conjugatedwith a bead for the cell. The samples after reverse transcription andreplication were PCR amplified for 15 cycles at 60 degrees annealingtemperature using primers for determining the mRNA expression profilesof 488 blood panel genes, using blood panel N1 primers, and theexpression of CD147 protein, using the sample indexing oligonucleotideN1 primers (“PCR 1”). Excess primers were removed with Ampure cleanup.The products from PCR1 were further PCR amplified (“PCR 2”) for 15cycles at 60 degrees annealing temperature using blood panel N2 primersand sample indexing oligonucleotide N1 primers with a flanking sequencefor adaptor ligation. Sequencing data was obtained and analyzed aftersequencing adaptor ligation (“PCR 3”).

FIGS. 38A-38D are non-limiting exemplary bioanalyzer traces showingpeaks (indicated by arrows) consistent with the expected size of theantibody oligonucleotide. The antibody oligonucleotide peaks decreasedas the hot antibody was titrated with the cold antibody.

Table 4 is a summary of sequencing data metrics. By staining the cellswith the mixture of 1% hot antibody:99% cold antibody prepared using the1:100 diluted stock, the antibody oligonucleotides accounted for 2.4% ofthe total raw reads in the sequencing data. However, as shown in FIGS.39A1-39A3 and 39B1-39B3 and FIGS. 40B, 41B, and 42B, a distributionhistogram of the numbers of molecules of antibody oligonucleotidesdetected after recursive substitution error correction (RSEC) ordistribution-based error correction (DBEC) did not include a clearsignal peak if the cells were stained with the mixture of 1% hotantibody:99% cold antibody prepared using the 1:100 diluted stock. RSEChas been described in U.S. patent application Ser. No. 15/605,874, filedon May 25, 2017, the content of which is incorporated herein byreference in its entirety.

FIGS. 40A-40C are non-limiting exemplary plots showing thatoligonucleotide-conjugated anti-CD147 antibody molecules can be used tolabel various cell types. The cell types were determined using theexpression profiles of 488 genes in a blood panel (FIG. 40A). The cellswere stained with a mixture of 10% hot antibody:90% cold antibodyprepared using a 1:100 diluted stock, resulting in a clear signal in ahistogram showing the numbers of molecules of antibody oligonucleotidesdetected (FIG. 40B). The labeling of the various cell types by theantibody oligonucleotide is shown in FIG. 40C.

FIGS. 41A-41C are non-limiting exemplary plots showing thatoligonucleotide-conjugated anti-CD147 antibodies can be used to labelvarious cell types. The cell types were determined using the expressionprofiles of 488 genes in a blood panel (FIG. 41A). The cells werestained with a mixture of 1% hot antibody:99% cold antibody preparedusing a 1:100 diluted stock, resulting in no clear signal in a histogramshowing the numbers of molecules of antibody oligonucleotides detected(FIG. 41B). The labeling of the various cell types by the antibodyoligonucleotide is shown in FIG. 41C.

FIGS. 42A-42C are non-limiting exemplary plots showing thatoligonucleotide-conjugated anti-CD147 antibody molecules can be used tolabel various cell types. The cell types were determined using theexpression profiles of 488 genes in a blood panel (FIG. 42A). The cellswere stained with a 1:800 diluted stock, resulting in a clear signal ina histogram showing the numbers of molecules of antibodyoligonucleotides detected (FIG. 42B). The labeling of the various celltypes by the antibody oligonucleotide is shown in FIG. 42C.

TABLE 4 Summary of sequencing data metrics. FC2 -1:100 FC4 - 1:800 FC3 -1:100 Dilution, Dilution, No Cold Dilution, 1:100 1:10 Cold SampleAntibody Cold Antibody Antibody Total Raw Reads 31.3M 27.3M 29.2M TotalRaw Reads 9161642 (29.2%) 660044 (2.4%) 4013438 Assigned to Oligos(13.7%) Cell Detected 1010 983 907 RSEC Oligo MI 577110 20054 170742DBEC Oligo MI 477216 9319 110629 % Q30 76.45 71.89 73.45 % assigned tocell 84.63 79.47 82.58 labels % aligned uniquely to 73.19 65.4 69.58amplicons Mean raw seq depth 5.58 6.52 6.4 Mean RSEC seq depth 8.6910.41 10.25 Mean DBEC seq depth 15.96 23.29 22.48 AbOligo RSEC seq 8.0812.9 11.2 depth AbOligo DBEC Seq 12.4 30.9 21.2 depth Mean reads percell 11257 14414 15150 mean molecules per 553.8 608.3 647.1 cell Medianmols per cell 246.5 279 278 No. of genes in panel 489 489 489 Totalgenes detected 438 439 441 Mean genes per cell 68.39 73.6 73.12

Altogether, these data indicate that the ratio ofoligonucleotide-conjugated antibodies (“hot antibodies”) and antibodiesnot conjugated with oligonucleotides (“cold antibody”) can be adjustedsuch that the antibody oligonucleotides account for a desired percentageof total reads in sequencing data and data representing signal antibodyoligonucleotides is clearly separated from data representing noiseantibody oligonucleotides.

Example 5 Normalization

This example demonstrates how normalization, using a mixture ofoligonucleotide-conjugated antibodies (“hot antibodies”) and antibodiesnot conjugated with oligonucleotides (“cold antibody”), can result inthe antibody oligonucleotides accounting for a desired percentage oftotal reads in sequencing data with a desired coverage, irrespective ofthe abundance of the protein targets of the antibodies.

Table 5 shows a comparison of quantification of three cell surfacemarkers of varying abundance in 10000 B cells using hot antibodies.Total number of reads required to resolve relative expression levels ofthe three cell surface markers was 47.52 million reads.

TABLE 5 Example protein quantification using hot antibodies. Number ofMolecules Number per Cell of reads Ratio of Detected given MoleculesRelative Hot:Cold by sequencing Antigen per Cell abundance AntibodiesSequencing depth of 4 CD21 210000 105 1:0 840 33.6M HLA- 85000 42.5 1:0340 13.6M DR CD40 2000 1 1:0 8  0.32M

TABLE 6 Example protein quantification using hot and cold antibodies.Number of Expected Molecules Number of number of per Cell readsmolecules Relative Ratio of detected given based on abundance MoleculesRelative Hot:Cold by sequencing Antibody by Antigen per Cell abundanceAntibodies sequencing depth of 4 ratio sequencing CD21 210000 105 1:1008.3 0.33M 8.3 × 100 = 830 103.75 HLA- 85000 42.5 1:40  8.5 0.34M 8.5 ×40 = 340 42.5 DR CD40 2000 1 1:0  8 0.32M 8 × 1 = 8 1

Table 6 shows that the total number of reads required to resolverelative expression levels of the three cell surface markers was 1million reads using mixtures of hot antibodies:cold antibodies. Also,only 2% of the number of reads, compared to the quantification resultshown in Table 5 (1 million reads vs. 47.52 million reads), is needed toachieve optimal coverage (e.g., sequencing depth of 4) of all threeprotein markers when mixtures of hot antibodies:cold antibodies wereused to quantify expression levels of the three cell surface markers.Normalizing high expressing protein molecules, using a mixture withhigher percentage of cold antibodies, decreased tradeoffs betweendetection of low abundance proteins, number of parameters, andsequencing cost, making the assay more attractive as a tool.

Altogether, these data indicate that a desired number of total reads insequencing data with a desired coverage can be achieved for proteintargets (e.g., antigens) of different abundance using mixtures of hotantibodies:cold antibodies.

Example 6 Control Particles

This example demonstrates generating control particles comprisingcontrol particle oligonucleotides with different sequences and use ofthe functionalized control particles to determine capture efficiency.

Materials

BD CompBead Plus Anti-Mouse Ig (7.5 um) Particles Set (51-9006274)

BD staining buffer (FBS)

Procedure

1. Vortex BD CompBead Plus thoroughly before use (1 minute at least).

2. Add 800 uL of staining buffer to tube (Table 7 shows the compositionof the staining buffer with CD147 conjugated to oligonucleotides withfive sequences at different abundance. FIG. 43A is a plot showing thecomposition of the staining buffer.).

3. Add 5 full drops (approximately 300 uL) of CompBead Plus Anti-Mouse.

4. Add 20 uL of the staining cocktail below to the tube. Vortex.

5. Incubate 30 minutes at room temperature away from light.

6. Spin beads at 200 g for 10 minutes.

7. Remove supernatant carefully and resuspend with 1 mL staining buffer.

8. Spin beads at 200 g for 10 minutes.

9. Remove supernatant carefully and resuspend with 1 mL staining bufferto generate the functionalized CompBead stock solution.

10. Count beads.

TABLE 7 Staining Cocktail Composition Final Prior Antibodies % inStaining Buffer Dilution Staining Solution (μl) CD147-LZ15 1 1:1 1CD147-LZ16 0.2 1:5 1 CD147-LZ17 0.1 1:10 1 CD147-LZ18 0.02 1:50 1CD147-LZ19 0.01 1:100 1 Staining Buffer 95

Results

FIGS. 44A-44B are brightfield images of cells (FIG. 44A, white circles)and control particles (FIG. 44B, black circles) in a hemocytometer.FIGS. 45A-45B are phase contrast (FIG. 45A, 10X) and fluorescent (FIG.45B, 10X) images of control particles bound tooligonucleotide-conjugated antibodies associated with fluorophores.Fluorescent microscope was used to determine that 5 ul of thefunctionalized CompBead stock solution contained ˜2000 cells (4% oftotal input) with ˜400000 functionalized CompBeads made.

FIG. 46 is an image of a control particle showing cells and a particlebeing loaded into microwells of a cartridge. CompBeads can be used withregular Rhapsody™ experiments. 522 functionalized CompBeads were addedinto a plurality of cells. Of the 20000 cells (including controlparticles) sequences, 156 had a sum of all control particleoligonucleotides greater than 20. Thus, 156 control particles weresequences. FIG. 43B is a plot showing the number of control particleoligonucleotides with the five different control barcode sequences(LZ15-LZ19) correlated with their abundance in the staining buffer.

Altogether, these data show that particles (e.g., CompBead Plus) can befunctionalized with oligonucleotides (e.g., control particleoligonucleotides). Functionalized particles can be used with single cellsequencing workflow to determine the number of particles captured andsequenced.

Example 7 Antibody Cocktail for Sample Indexing

This example demonstrates using an antibody cocktail for sample indexingcan increase labeling sensitivity.

Sample indexing can utilize oligonucleotide-conjugated antibodiesagainst multiple protein targets to label samples. In this example,instead of using a single antibody, a cocktail of two antibodies wereused instead. The cocktail of antibodies were labeled with the samesample indexing oligonucleotides. FIGS. 47A-47C are plots showing usingan antibody cocktail for sample indexing can increase labelingsensitivity. FIGS. 47A-47C show the labeling sensitivity of PBMCs with aCD147 antibody, a CD47 antibody, and both CD147 and CD47 antibodies.FIG. 47C shows that more cells were labeled when both CD147 and CD47antibodies were used and better signal to noise separation was achieved.Table 8 shows increased labeling sensitivity when both CD147 and CD47antibodies were used. The use of both CD147 and CD47 antibodies resultedin higher sensitivity for labeling heterogeneous sample types.

TABLE 8 Labeling sensitivity with CD147, CD47, or both CD147 and CD47antibodies. Antibody Sensitivity CD147 86.2% CD47 82.5% CD147 + CD4792.6%

Altogether, the data show that using an antibody cocktail can increaselabeling sensitivity. This may be because protein expression can varybetween cell types and cell states, making finding a universal antibodythat labels all cell types challenging. Using an antibody cocktail canallow for more sensitive and efficient labeling of more sample types. Anantibody cocktail can also include a wider range of antibodies.Antibodies that label different sample types well can be pooled togetherto create a cocktail that sufficiently labels all cell types or celltypes of interest.

Example 8 Presence of Multplets in Datasets

This example describes that multiplets in sequencing data can beidentified and removed by tagging cells with different sample indexingoligonucleotides.

FIG. 48 is a non-limiting exemplary plot showing that multiplets can beidentified and removed from sequencing data using sample indexing. Asshown in FIG. 48, the rate of multiplets in droplets and single cellexpression data can be close to eight percent when a sample with single20000 cells are partitioned into droplets using a droplet-basedplatform. The rate of multiplets in a microwell array and single cellexpression data can be much lower (e.g., close to only four percent)when a sample with single 20000 cells are partitioned into wells of aRhapsody™ (Becton, Dickinson and Company (Franklin Lakes, N.J.)).Multiplets in sequencing data can be identified and removed using sampleindexing oligonucleotides with different sample indexing sequences. Forexample, when a sample with 20000 single cells is divided into twosubpopulations, cells in each subpopulation are labeled with sampleindexing oligonucleotides with an identical sample indexing sequence,and cells in different subpopulations are labeled with sample indexingoligonucleotides with two different sample indexing sequences, sequencedata associated with multiplets can be identified and removed, with theresulting sequencing data containing around two % multiplets. Withsample indexing oligonucleotides with eight different sample indexingsequences, sequencing data of 20000 single cells can include only around0.5% multiplets. With a higher number of sample indexing sequences, therate of multiplet in sequencing data can be even lower.

Thus, the rate of multiplets remaining in single cell sequencing datacan be substantially lowered, or eliminated, by labeling cells withsample indexing oligonucleotides with different sample indexingsequences.

Example 9 Sample Indexing for Identifying Multiplets

This example demonstrates using sample tags to identify and removemultiplets in sequencing data.

Six tissues (bone marrow, fat (gonadal white adipose tissue (gWAT)),colon, liver, lung, and spleen) from two mice were obtained. CD45+single cells from the isolated tissues were isolated and sorted usingfluorescence-activated cell sorting (FACS) to create 12 samples. CD45+single cells of the 12 samples were tagged with 12 different sample tags(referred to herein as sample indexing compositions) using BD™Single-Cell Multiplexing Kit for RNA-Sequencing and loaded onto aRhapsody™ cartridge. A sample tag was an antibody conjugated to anoligonucleotide (referred to herein as sample indexing oligonucleotides)that can be captured and amplified in a 3′ RNA-seq assay (such as the BDRhapsody™ assay). Such a sample tag had high specificity and sensitivity(>99%). The mRNA molecules from the cells and the sample indexingoligonucleotides were captured using Rhapsody™ magnetic beads, andbarcoded and amplified using Rhapsody™ Immune Response Panel-Mouse(Mouse). Expression profiles of the cells were determined and identifiedas singlet expression profiles and multiplet expression profiles usingsynthetic multiplet expression profiles.

FIG. 49 is a non-limiting exemplary tSNE projection plot of expressionprofiles of CD45+ single cells from 12 samples of six tissues from twomice identified as singlets or multiplets using sample tags. FIG. 49shows the multiplet expression profiles as multiple clusters. Sample tagsequences (also referred to herein as sample indexing indices of sampleindexing oligonucleotides) for tagging the different tissues were usedto identify multiplet expression profiles each including the expressionprofiles of cells of two or more cell types or subtypes. Multiplets ofCD45+ cells from two different tissues were identified based on thepresence of the sample tag sequences in the sequence data obtained fromthe cells. The performance of using nucleic acid sample tags to indexsamples and identify multiplets was comparable to the performance ofusing synthetic multiplet expression profiles to identify multiplets.FIG. 49B is a non-limiting exemplary tSNE projection plot of expressionprofiles of CD45+ single cells of 12 samples of six tissues from twomice with multiplets identified using sample indexing oligonucleotidesremoved.

FIG. 50A is a non-limiting exemplary tSNE projection plot of expressionprofiles of CD45+ single cells from two mice with multiplets identifiedusing sample indexing oligonucleotides removed. With multipletexpression profiles removed, the two mice, which were biologicalreplicates, exhibited similar expression profiles as shown in FIGS.50A-50C. The results of immune profiling of the six different tissuesafter removing multiplet expression profiles are shown in FIGS. 51A-51F.The different cell types (e.g., B cells, macrophages, etc.) in the sixsample shown in FIGS. 51A-51F were identified based on the expressionprofiles of the cells.

Exemplary expression profiles of macrophages, T cells, and B cells fromthe six different tissues are shown in FIGS. 52A-52C. FIGS. 52A-52C showthat the same cell types in different tissues exhibited uniqueexpression profiles and colon immune populations were the most distinctout of the six tissues. FIGS. 53A-53D are non-limiting exemplary plotscomparing macrophages in the colon and spleen. FIG. 53A is anon-limiting exemplary tSNE projection plot comparing the expressionprofiles of macrophages in the colon and spleen, showing thatmacrophages in the two tissues had distinct expression profiles. FIG.53B is a non-limiting exemplary plot showing the expression profiles ofmacrophages in the colon and spleen had low correlation. The lowcorrelation further supported that macrophages in the two tissues haddistinct expression profiles shown in FIG. 53A. FIGS. 53C and 53D arenon-limiting exemplary tSNE projection plots of expression profiles ofthe Areg gene and Rora gene in macrophages in the colon and spleen.These two genes were expressed in 5.2% and 9.5% of the macrophages inthe colon and spleen. FIGS. 53A, 53C, and 53D show that these two geneswere expressed only in macrophages in the colon. Single-cell profilingof more than 28000 mouse immune cells from six different tissues asshown in FIGS. 52A-52C and 53A-53D revealed tissue specific geneexpression signatures for major immune cell types.

Altogether, these data show that sample tagging can allow pooling ofmultiple samples onto the same single-cell experiment. Sample Taggingcan increase sample throughput, eliminate batch effect between samplesdue to technical errors and enable detection of multiplets.

Example 10 Sample Indexing and Expression Profiles

This example demonstrates using sample tagging of cells does not altermRNA expression profiles of cells.

FIG. 54 is a non-limiting exemplary plot showing that tagging orstaining cells with sample indexing compositions did not alter mRNAexpression profiles in peripheral blood mononuclear cell (PBMCs). FIG.54 shows an overlay of tSNE projection plots of expression profiles ofPBMCs with or without sample tagging. The overlap shows that sampletagging did not alter mRNA expression profiles in PBMCs.

Altogether, the data show that sample tagging or staining withantibodies conjugated with oligonucleotides do not alter expressionprofiles of cells. Thus, sample tagging can allow pooling of multiplesamples, increase sample throughput, eliminate batch effect betweensamples, and/or enable detection of multiplets without altering theexpression profiles of the tagged cells.

Terminology

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described above without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. As used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise. Any reference to “or” herein isintended to encompass “and/or” unless otherwise stated.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

1.-4. (canceled)
 5. A method for sample identification, comprising:contacting one or more cells from each of a plurality of samples with asample indexing composition of a plurality of sample indexingcompositions, wherein each of the one or more cells comprises one ormore cellular component targets, wherein at least one sample indexingcomposition of the plurality of sample indexing compositions comprises acellular component binding reagent, wherein the cellular componentbinding reagent is associated with a sample indexing oligonucleotide,wherein at least one of the two or more the cellular component bindingreagent is capable of specifically binding to at least one of the one ormore cellular component targets, wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of at least two sample indexing compositions of theplurality of sample indexing compositions comprise different sequences;and identifying sample origin of at least one cell of the one or morecells based on the sample indexing sequence of at least one sampleindexing oligonucleotide of the plurality of sample indexingcompositions.
 6. The method of claim 5, wherein the cellular componentbinding reagent comprises a protein binding reagent, wherein the proteinbinding reagent is associated with the sample indexing oligonucleotideassociated with the corresponding cellular component binding reagent,and wherein the cellular component target comprises a protein target. 7.The method of claim 5, wherein identifying the sample origin of the atleast one cell comprises: barcoding sample indexing oligonucleotides ofthe plurality of sample indexing compositions using a plurality ofbarcodes to create a plurality of barcoded sample indexingoligonucleotides; obtaining sequencing data of the plurality of barcodedsample indexing oligonucleotides; and identifying the sample origin ofthe cell based on the sample indexing sequence of at least one barcodedsample indexing oligonucleotide of the plurality of barcoded sampleindexing oligonucleotides in the sequencing data.
 8. The method of claim5, wherein identifying the sample origin of the at least one cellcomprises identifying the presence or absence of the sample indexingsequence of at least one sample indexing oligonucleotide of theplurality of sample indexing compositions.
 9. The method of claim 8,wherein identifying the presence or absence of the sample indexingsequence comprises: replicating the at least one sample indexingoligonucleotide to generate a plurality of replicated sample indexingoligonucleotides; obtaining sequencing data of the plurality ofreplicated sample indexing oligonucleotides; and identifying the sampleorigin of the cell based on the sample indexing sequence of a replicatedsample indexing oligonucleotide of the plurality of sample indexingoligonucleotides that correspond to the least one barcoded sampleindexing oligonucleotide in the sequencing data.
 10. The method of claim9, wherein replicating the at least one sample indexing oligonucleotideto generate the plurality of replicated sample indexing oligonucleotidescomprises: prior to replicating the at least one barcoded sampleindexing oligonucleotide, ligating a replicating adaptor to the at leastone barcoded sample indexing oligonucleotide, and wherein replicatingthe at least one barcoded sample indexing oligonucleotide comprisesreplicating the at least one barcoded sample indexing oligonucleotideusing the replicating adaptor ligated to the at least one barcodedsample indexing oligonucleotide to generate the plurality of replicatedsample indexing oligonucleotides.
 11. The method of claim 9, whereinreplicating the at least one sample indexing oligonucleotide to generatethe plurality of replicated sample indexing oligonucleotides comprises:prior to replicating the at least one barcoded sample indexingoligonucleotide, contacting a capture probe with the at least one sampleindexing oligonucleotide to generate a capture probe hybridized to thesample indexing oligonucleotide; and extending the capture probehybridized to the sample indexing oligonucleotide to generate a sampleindexing oligonucleotide associated with the capture probe, and whereinreplicating the at least one sample indexing oligonucleotide comprisesreplicating the sample indexing oligonucleotide associated with thecapture probe to generate the plurality of replicated sample indexingoligonucleotides.
 12. The method of claim 5, wherein the sample indexingsequence is 6-60 nucleotides in length, and wherein the sample indexingoligonucleotide is 50-500 nucleotides in length.
 13. (canceled)
 14. Themethod of claim 5, wherein sample indexing sequences of at least 10sample indexing compositions of the plurality of sample indexingcompositions comprise different sequences.
 15. (canceled)
 16. (canceled)17. (canceled)
 18. The method of claim 5 wherein the cellular componenttargets is on a cell surface.
 19. The method of claim 5, comprisingremoving unbound sample indexing compositions of the plurality of sampleindexing compositions.
 20. (canceled)
 21. (canceled)
 22. (canceled) 23.The method of claim 5, wherein the sample indexing oligonucleotide isconfigured to be non-detachable from the cellular component bindingreagent.
 24. The method of claim 5, comprising detaching the sampleindexing oligonucleotide from the cellular component binding reagent.25. (canceled)
 26. (canceled)
 27. (canceled)
 28. (canceled) 29.(canceled)
 30. The method of claim 5, wherein the sample indexingoligonucleotide comprises a sequence complementary to a capture sequenceconfigured to capture the sequence of the sample indexingoligonucleotide, and wherein the barcode comprises a target-bindingregion which comprises the capture sequence.
 31. (canceled)
 32. Themethod of claim 30, wherein the target-binding region comprises apoly(dT) region, and wherein the sequence of the sample indexingoligonucleotide complementary to the capture sequence comprises apoly(dA) region.
 33. (canceled)
 34. The method of claim 1, wherein thesample indexing oligonucleotide comprises a molecular label sequence, abinding site for a universal primer, or both.
 35. The method of claim 1wherein the cellular component target comprises a carbohydrate, a lipid,a protein, an extracellular protein, a cell-surface protein, a cellmarker, a B-cell receptor, a T-cell receptor, a major histocompatibilitycomplex, a tumor antigen, a receptor, an intracellular protein or anycombination thereof.
 36. (canceled)
 37. (canceled)
 38. (canceled) 39.(canceled)
 40. (canceled)
 41. (canceled)
 42. (canceled)
 43. (canceled)44. The method of claim 5, wherein each of the plurality of sampleindexing compositions comprises the cellular component binding reagent.45. (canceled)
 46. (canceled)
 47. (canceled)
 48. The method of claim 7,wherein the plurality of barcodes is associated with a particle.
 49. Themethod of claim 48, wherein at least one barcode of the plurality ofbarcodes is immobilized on the particle, partially immobilized on theparticle, enclosed in the particle, partially enclosed in the particle,or a combination thereof.
 50. The method of claim 48, wherein theparticle is a disruptable bead or a disruptable hydrogel bead. 51.(canceled)
 52. (canceled)
 53. (canceled)
 54. (canceled)
 55. (canceled)56. (canceled)
 57. The method of claim 48, wherein barcoding the sampleindexing oligonucleotides using the plurality of barcodes comprises:contacting the plurality of barcodes with the sample indexingoligonucleotides to generate barcodes hybridized to the sample indexingoligonucleotides; and extending the barcodes hybridized to the sampleindexing oligonucleotides to generate the plurality of barcoded sampleindexing oligonucleotides.
 58. (canceled)
 59. (canceled)
 60. (canceled)61. (canceled)
 62. (canceled)
 63. (canceled)
 64. (canceled)
 65. Themethod of claim 5, comprising: barcoding a plurality of targets of thecell using the plurality of barcodes to create a plurality of barcodedtargets, wherein each of the plurality of barcodes comprises a celllabel sequence, and wherein at least two barcodes of the plurality ofbarcodes comprise an identical cell label sequence; and obtainingsequencing data of the barcoded targets.
 66. (canceled)
 67. (canceled)68. (canceled)
 69. (canceled)
 70. A plurality of sample indexingcompositions, wherein each of the plurality of sample indexingcompositions comprises a cellular component binding reagent, wherein thecellular component binding reagent is associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one cellular componenttarget, wherein the sample indexing oligonucleotide comprises a sampleindexing sequence for identifying sample origin of one or more cells ofa sample, and wherein sample indexing sequences of at least two sampleindexing compositions of the plurality of sample indexing compositionscomprise different sequences.
 71. (canceled)
 72. (canceled) 73.(canceled)
 74. The plurality of sample indexing compositions of claim70, wherein the cellular component binding reagent comprises anantibody, a tetramer, an aptamer, a protein scaffold, or a combinationthereof.
 75. The plurality of sample indexing compositions of claim 70,wherein the sample indexing oligonucleotide is conjugated to thecellular component binding reagent through a linker.
 76. (canceled) 77.(canceled)
 78. (canceled)
 79. (canceled)
 80. (canceled)
 81. (canceled)82. The plurality of sample indexing compositions of claim 70, whereinthe cellular component binding reagent is associated with two or moresample indexing oligonucleotides with an identical sequence.
 83. Theplurality of sample indexing compositions of claim 70, wherein thecellular component binding reagent is associated with two or more sampleindexing oligonucleotides with different sample indexing sequences. 84.The plurality of sample indexing compositions of claim 70, wherein thesample indexing composition of the plurality of sample indexingcompositions comprises a second cellular component binding reagent notassociated with the sample indexing oligonucleotide.
 85. The pluralityof sample indexing compositions of claim 84, where the cellularcomponent binding reagent and the second cellular component bindingreagent are identical. 86.-595. (canceled)